Immunomodulators

ABSTRACT

The present disclosure provides novel macrocyclic peptides which inhibit the PD-1/PD-L1 and PD-L1/CD80 protein/protein interaction, and thus are useful for the amelioration of various diseases, including cancer and infectious diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/094,008 filed Dec. 18, 2014 hereby incorporated by reference inits entirety.

The present disclosure provides novel macrocyclic peptides which inhibitthe PD-1/PD-L1 and CD80/PD-L1 protein/protein interaction, and are thususeful for the amelioration of various diseases, including cancer andinfectious diseases.

The protein Programmed Death 1 (PD-1) is an inhibitory member of theCD28 family of receptors, that also includes CD28, CTLA-4, ICOS andBTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells(Agata et al., supra; Okazaki et al., Curr. Opin. Immunol., 14:779-782(2002); Bennett et al., J. Immunol., 170:711-718 (2003)).

The PD-1 protein is a 55 kDa type I transmembrane protein that is partof the Ig gene superfamily (Agata et al., Int. Immunol., 8:765-772(1996)). PD-1 contains a membrane proximal immunoreceptor tyrosineinhibitory motif (ITIM) and a membrane distal tyrosine-based switchmotif (ITSM) (Thomas, M. L., J. Exp. Med., 181:1953-1956 (1995); Vivier,E. et al., Immunol. Today, 18:286-291 (1997)). Although structurallysimilar to CTLA-4, PD-1 lacks the MYPPY motif that is critical for CD80CD86 (B7-2) binding. Two ligands for PD-1 have been identified, PD-L1(B7-H1) and PD-L2 (b7-DC). The activation of T cells expressing PD-1 hasbeen shown to be downregulated upon interaction with cells expressingPD-L1 or PD-L2 (Freeman et al., J. Exp. Med., 192:1027-1034 (2000);Latchman et al., Nat. Immunol., 2:261-268 (2001); Carter et al., Eur. J.Immunol., 32:634-643 (2002)). Both PD-L1 and PD-L2 are B7 protein familymembers that bind to PD-1, but do not bind to other CD28 family members.The PD-L1 ligand is abundant in a variety of human cancers (Dong et al.,Nat. Med., 8:787-789 (2002)). The interaction between PD-1 and PD-L1results in a decrease in tumor infiltrating lymphocytes, a decrease inT-cell receptor mediated proliferation, and immune evasion by thecancerous cells (Dong et al., J. Mol. Med., 81:281-287 (2003); Blank etal., Cancer Immunol. Immunother., 54:307-314 (2005); Konishi et al.,Clin. Cancer Res., 10:5094-5100 (2004)). Immune suppression can bereversed by inhibiting the local interaction of PD-1 with PD-L1, and theeffect is additive when the interaction of PD-1 with PD-L2 is blocked aswell (Iwai et al., Proc. Natl. Acad. Sci. USA, 99:12293-12297 (2002);Brown et al., J. Immunol., 170:1257-1266 (2003)).

PD-L1 has also been shown to interact with CD80 (Butte M J et al,Immunity; 27:111-122 (2007)). The interaction PD-L1/CD80 on expressingimmune cells has been shown to be an inhibitory one. Blockade of thisinteraction has been shown to abrogate this inhibitory interaction(Paterson A M, et al., J Immunol., 187:1097-1105 (2011); Yang J, et al.J Immunol. August 1; 187(3):1113-9 (2011)).

When PD-1 expressing T cells contact cells expressing its ligands,functional activities in response to antigenic stimuli, includingproliferation, cytokine secretion, and cytotoxicity, are reduced.PD-1/PD-L1 or PD-L2 interactions down regulate immune responses duringresolution of an infection or tumor, or during the development of selftolerance (Keir, M. E. et al., Annu. Rev. Immunol., 26:Epub (2008)).Chronic antigen stimulation, such as that which occurs during tumordisease or chronic infections, results in T cells that express elevatedlevels of PD-1 and are dysfunctional with respect to activity towardsthe chronic antigen (reviewed in Kim et al., Curr. Opin. Imm. (2010)).This is termed “T cell exhaustion”. B cells also display PD-1/PD-ligandsuppression and “exhaustion”.

Blockade of PD-1/PD-L1 ligation using antibodies to PD-L1 has been shownto restore and augment T cell activation in many systems. Patients withadvanced cancer benefit from therapy with a monoclonal antibody to PD-L1(Brahmer et al., New Engl. J. Med. (2012)). Preclinical animal models oftumors and chronic infections have shown that blockade of the PD-1/PD-L1pathway by monoclonal antibodies can enhance the immune response andresult in tumor rejection or control of infection. Antitumorimmunotherapy via PD-1/PD-L1 blockade may augment therapeutic immuneresponse to a number of histologically distinct tumors (Dong, H. et al.,“B7-H1 pathway and its role in the evasion of tumor immunity”, J. Mol.Med., 81(5):281-287 (2003); Dong, H. et al., “Tumor-associated B7-H1promotes T-cell apoptosis: a potential mechanism of immune evasion”,Nat. Med., 8(8):793-800 (2002)).

Interference with the PD-1/PD-L1 interaction causes enhanced T cellactivity in systems with chronic infection. Blockade of PD-L1 causedimproved viral clearance and restored immunity in mice with chromoiclymphocytic chorio meningitis virus infection (Barber, D. L. et al.,“Restoring function in exhausted CD8 T cells during chronic viralinfection”, Nature, 439(7077):682-687 (2006)). Humanized mice infectedwith HIV-1 show enhanced protection against viremia and viral depletionof CD4+ T cells (Palmer et al., J. Immunol. (2013)). Blockade ofPD-1/PD-L1 through monoclonal antibodies to PD-L1 can restore in vitroantigen-specific functionality to T cells from HIV patients (Day, Nature(2006); Petrovas, J. Exp. Med. (2006); Trautman, Nature Med. (2006);D'Souza, J. Immunol. (2007); Zhang, Blood (2007); Kaufmann, Nature Imm.(2007); Kasu, J. Immunol. (2010); Porichis, Blood (2011)), HCV patients(Golden-Mason, J. Virol. (2007); Jeung, J. Leuk. Biol. (2007); Urbani,J. Hepatol. (2008); Nakamoto, PLoS Path. (2009); Nakamoto,Gastroenterology (2008)) and HBV patients (Boni, J. Virol. (2007);Fisicaro, Gastro. (2010); Fisicaro et al., Gastroenterology (2012); Boniet al., Gastro. (2012); Penna et al., J. Hep. (2012); Raziorrough,Hepatology (2009); Liang, World J. Gastro. (2010); Zhang, Gastro.(2008)).

Blockade of the PD-L1/CD80 interaction has also been shown to stimulateimmunity (Yang J., et al., J. Immunol. August 1; 187(3):1113-9 (2011)).Immune stimulation resulting from blockade of the PD-L1/CD80 interactionhas been shown to be enhanced through combination with blockade offurther PD-1/PD-L1 or PD-1/PD-L2 interactions.

Alterations in immune cell phenotypes are hypothesized to be animportant factor in septic shock (Hotchkiss, et al., Nat Rev Immunol(2013)). These include increased levels of PD-1 and PD-L1 (Guignant, etal, Crit. Care (2011)), Cells from septic shock patients with increasedlevels of PD-1 and PD-L1 exhibit an increased level of T cell apoptosis.Antibodies directed to PD-L1, can reduce the level of Immune cellapoptosis (Zhang et al, Crit. Care (2011)). Furthermore, mice lackingPD-1 expression are more resistant to septic shock symptoms thanwildtype mice. Yang J., et al. J Immunol. August 1; 187(3):1113-9(2011)). Studies have revealed that blockade of the interactions ofPD-L1 using antibodies can suppress inappropriate immune responses andameliorate disease signs.

In addition to enhancing immunologic responses to chronic antigens,blockade of the PD-1/PD-L1 pathway has also been shown to enhanceresponses to vaccination, including therapeutic vaccination in thecontext of chronic infection (Ha, S. J. et al., “Enhancing therapeuticvaccination by blocking PD-1-mediated inhibitory signals during chronicinfection”, J. Exp. Med., 205(3):543-555 (2008); Finnefrock, A. C. etal., “PD-1 blockade in rhesus macaques: impact on chronic infection andprophylactic vaccination”, J. Immunol., 182(2):980-987 (2009); Song,M.-Y. et al., “Enhancement of vaccine-induced primary and memoryCD8+t-cell responses by soluble PD-1”, J. Immunother., 34(3):297-306(2011)).

The molecules described herein demonstrate the ability to block theinteraction of PD-L1 with PD-1, in both biochemical and cell-basedexperimental systems. These results are consistent with a potential fortherapeutic administration to enhance immunity in cancer or chronicinfection, including therapeutic vaccine.

The macrocyclic peptides described herein are capable of inhibiting theinteraction of PD-L1 with PD-1 and with CD80. These compounds havedemonstrated highly efficacious binding to PD-L1, blockade of theinteraction of PD-L1 with either PD-1 or CD80, and are capable ofpromoting enhanced T cell functional activity, thus making themcandidates for parenteral, oral, pulmonary, nasal, buccal and sustainedrelease formulations.

In one aspect the present disclosure provides a compound of formula (I).

or a pharmaceutically acceptable salt thereof, wherein:

-   -   A is selected from a bond,

wherein:

-   -   denotes the point of attachment to the carbonyl group and        denotes the point of attachment to the nitrogen atom;    -   z is 0, 1, or 2;    -   w is 1 or 2;    -   n is 0 or 1;    -   m is 1 or 2;    -   m′ is 0 or 1;    -   p is 0, 1, or 2;    -   R^(x) is selected from hydrogen, amino, hydroxy, and methyl;    -   R¹⁴ and R¹⁵ are independently selected from hydrogen and methyl;        and    -   R^(z) is selected from hydrogen and —C(O)NHR¹⁶; wherein R¹⁶ is        selected from hydrogen, —CHR¹⁷C(O)NH₂, —CHR¹⁷C(O)NHCHR¹⁸C(O)NH₂,        and —CHR¹⁷C(O)NHCHR¹⁸C(O)NHCH₂C(O)NH₂; wherein R¹⁷ is selected        from hydrogen and —CH₂OH and wherein R¹⁸ is selected from        hydrogen and methyl;    -   R^(v) is hydrogen or a natural amino acid side chain;    -   R^(c), R^(f), R^(h), R^(i), and R^(m) are hydrogen;    -   R^(n) is hydrogen or methyl or R^(v) and R^(n) form a        pyrrolidine ring;    -   R^(a) is hydrogen or methyl;    -   R^(j) is selected from hydrogen, C₁-C₆alkoxyC₁-C₆alkyl,        C₁-C₆alkyl, carboxyC₁-C₆alkyl, haloC₁-C₆alkyl,        hydroxyC₁-C₆alkyl, (NR^(a′)R^(b′))C₁-C₆alkyl wherein R^(a′) and        R^(b′) are independently selected from hydrogen and C₁-C₆alkyl;        and phenylC₁-C₆alkyl wherein the phenyl is optionally        substituted with one, two, three, four or five groups        independently selected from C₁-C₆alkoxy, C₁-C₆alkyl, cyano,        halo, and nitro;    -   R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², and R¹³ are        independently selected from a natural amino acid side chain and        an unnatural amino acid side chain; or    -   R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², and R¹³ can        each independently form a ring with the corresponding vicinal R        group as described below;    -   R^(b) is selected from C₁-C₆alkoxyC₁-C₆alkyl, C₁-C₆alkyl,        carboxyC₁-C₆alkyl, haloC₁-C₆alkyl, hydroxyC₁-C₆alkyl,        (NR^(a′)R^(b′))C₁-C₆alkyl wherein R^(a′) and R^(b′) are        independently selected from hydrogen and C₁-C₆alkyl; and        phenylC₁-C₆alkyl wherein the phenyl is optionally substituted        with one, two, three, four or five groups independently selected        from C₁-C₆alkoxy, C₁-C₆alkyl, cyano, halo, and nitro; or, R^(b)        and R², together with the atoms to which they are attached, form        a ring selected from azetidine, pyrolidine, morpholine,        piperidine, piperazine, and tetrahydrothiazole; wherein each        ring is optionally substituted with one to four groups        independently selected from amino, cyano, methyl, halo, and        hydroxy;    -   R^(d) is selected from hydrogen, C₁-C₆alkoxyC₁-C₆alkyl,        C₁-C₆alkyl, carboxyC₁-C₆alkyl, haloC₁-C₆alkyl,        hydroxyC₁-C₆alkyl, (NR^(a′)R^(b′))C₁-C₆alkyl wherein R^(a′) and        R^(b′) are independently selected from hydrogen and C₁-C₆alkyl;        and phenylC₁-C₆alkyl wherein the phenyl is optionally        substituted with one, two, three, four or five groups        independently selected from C₁-C₆alkoxy, C₁-C₆alkyl, cyano,        halo, and nitro; or, R^(d) and R⁴, together with the atoms to        which they are attached, can form a ring selected from        azetidine, pyrolidine, morpholine, piperidine, piperazine, and        tetrahydrothiazole; wherein each ring is optionally substituted        with one to four groups independently selected from amino,        cyano, methyl, halo, hydroxy, and phenyl;    -   R^(e) is selected from hydrogen, C₁-C₆alkoxyC₁-C₆alkyl,        C₁-C₆alkyl, carboxyC₁-C₆alkyl, haloC₁-C₆alkyl,        hydroxyC₁-C₆alkyl, (NR^(a′)R^(b′))C₁-C₆alkyl wherein R^(a′) and        R^(b′) are independently selected from hydrogen and C₁-C₆alkyl;        and phenylC₁-C₆alkyl wherein the phenyl is optionally        substituted with one, two, three, four or five groups        independently selected from C₁-C₆alkoxy, C₁-C₆alkyl, cyano,        halo, and nitro; or R^(e) and R⁵′ together with the atoms to        which they are attached, can form a ring selected from        azetidine, pyrolidine, morpholine, piperidine, piperazine, and        tetrahydrothiazole; wherein each ring is optionally substituted        with one to four groups independently selected from amino,        cyano, methyl, halo, and hydroxy;    -   R^(g) is selected from hydrogen, C₁-C₆alkoxyC₁-C₆alkyl,        C₁-C₆alkyl, carboxyC₁-C₆alkyl, haloC₁-C₆alkyl,        hydroxyC₁-C₆alkyl, (NR^(a′)R^(b′))C₁-C₆alkyl wherein R^(a′) and        R^(b′) are independently selected from hydrogen and C₁-C₆alkyl;        and phenylC₁-C₆alkyl wherein the phenyl is optionally        substituted with one, two, three, four or five groups        independently selected from C₁-C₆alkoxy, C₁-C₆alkyl, cyano,        halo, and nitro; or R^(g) and R⁷, together with the atoms to        which they are attached, can form a ring selected from        azetidine, pyrolidine, morpholine, piperidine, piperazine, and        tetrahydrothiazole; wherein each ring is optionally substituted        with one to four groups independently selected from amino,        benzyl optionally substituted with a halo group, benzyloxy,        cyano, cyclohexyl, methyl, halo, hydroxy, isoquinolinyloxy        optionally substituted with a methoxy group, quinolinyloxy        optionally substituted with a halo group, and tetrazolyl; and        wherein the pyrrolidine and the piperidine ring are optionally        fused to a cyclohexyl, phenyl, or indole group;    -   provided that at least one of R^(a), R^(b), R^(d), R^(e), R^(g),        R^(j), R^(k), and R^(l) is selected from, C₁-C₆alkoxyC₁-C₆alkyl,        C₂-C₆alkyl, carboxyC₁-C₆alkyl, haloC₁-C₆alkyl,        hydroxyC₁-C₆alkyl, (NR^(a′)R^(b′))C₁-C₆alkyl wherein IV and        R^(b′) are independently selected from hydrogen and C₁-C₆alkyl;        and phenylC₁-C₆alkyl wherein the phenyl is optionally        substituted with one, two, three, four or five groups        independently selected from C₁-C₆alkoxy, C₁-C₆alkyl, cyano,        halo, and nitro.

In a first embodiment of the first aspect the present disclosureprovides a compound of formula (I), or a pharmaceutically acceptablesalt thereof, wherein R^(e) is selected from hydrogen and methyl, orR^(e) and R⁵′ together with the atoms to which they are attached, canform a ring selected from azetidine, pyrolidine, morpholine, piperidine,piperazine, and tetrahydrothiazole; wherein each ring is optionallysubstituted with one to four groups independently selected from amino,cyano, methyl, halo, and hydroxy; and R^(j) is hydrogen or methyl. In asecond embodiment of the first aspect the present disclosure R^(k) isselected from hydrogen and methyl, or R^(k) and R¹¹, together with theatoms to which they are attached, can form a ring selected fromazetidine, pyrolidine, morpholine, piperidine, piperazine, andtetrahydrothiazole; wherein each ring is optionally substituted with oneto four groups independently selected from amino, cyano, methyl, halo,and hydroxy; and R¹ is methyl, or, R¹ and R¹², together with the atomsto which they are attached, form a ring selected from azetidine andpyrolidine, wherein each ring is optionally substituted with one to fourgroups independently selected from amino, cyano, methyl, halo, andhydroxy. In a third aspect A is

wherein

-   -   z and w are each 1;    -   R¹⁴ and R¹⁵ are hydrogen; and    -   R^(z) is —C(O)NHR¹⁶; wherein R¹⁶ is —CHR¹⁷C(O)NH₂;    -   R¹⁷ is hydrogen;    -   R¹ is benzyl optionally substituted with hydroxy;    -   R² is hydrogen or methyl;    -   R⁸ is —(CH₂)indolyl;    -   R¹⁰ is selected from —(CH₂)indolyl and —(CH₂)benzothienyl, each        optionally substituted with —CH₂CO₂H;    -   R¹¹ is butyl; and    -   R¹² is butyl.

In another embodiment the present disclosure provides a method ofenhancing, stimulating, and/or increasing the immune response in asubject in need thereof, said method comprising administering to thesubject a therapeutically effective amount of a compound of formula (I)or a therapeutically acceptable salt thereof. In another embodiment themethod further comprises administering an additional agent prior to,after, or simultaneously with the compound of formula (I) or atherapeutically acceptable salt thereof. In another embodiment theadditional agent is an antimicrobial agent, an antiviral agent, acytotoxic agent, and/or an immune response modifier. In anotherembodiment the additional agent is an HDAC inhibitor. In anotherembodiment the additional agent is a TLR7 and/or TLR8 agonist.

In another embodiment the present disclosure provides a method ofinhibiting growth, proliferation, or metastasis of cancer cells in asubject in need thereof, said method comprising administering to thesubject a therapeutically effective amount of a compound of formula (I)or a therapeutically acceptable salt thereof. It should be understoodthat said inhibition can be direct or indirect. In another embodimentthe cancer is selected from melanoma, renal cell carcinoma, squamousnon-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectalcancer, castration-resistant prostate cancer, ovarian cancer, gastriccancer, hepatocellular carcinoma, pancreatic carcinoma, squamous cellcarcinoma of the head and neck, carcinomas of the esophagus,gastrointestinal tract and breast, and a hematological malignancy.

In another embodiment the present disclosure provides a method oftreating an infectious disease in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of a compound of formula (I) or a therapeutically acceptable saltthereof. In another embodiment the infectious disease is caused by avirus. In another embodiment the virus is selected from HIV, HepatitisA, Hepatitis B, Hepatitis C, herpes virus, and influenza.

In another embodiment the present disclosure provides a method oftreating septic shock in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of one or more macrocyclic peptides described herein.

In another embodiment the present disclosure provides a method blockingthe interaction of PD-L1 with PD-1 and/or CD80 in a subject, said methodcomprising administering to the subject a therapeutically effectiveamount of at least one macrocyclic peptide described herein.

In compounds of formula (I) where the R side chains are part of a ringthat is substituted with methyl, it is understood that the methyl groupmay be on any substitutable carbon atom in the ring, including thecarbon that is part of the macrocyclic parent structure.

In compounds of formula (I), preferred R¹ side chains are:phenylalanine, tyrosine, 3-thien-2-yl, 4-methylphenylalanine,4-chlorophenylalanine, 3-methoxyphenylalananie, isotryptophan,3-methylphenylalanine, 1-naphthylalanine, 3,4-difluorophenylalanine,4-fluorophenylalanine, 3,4-dimethoxyphenylalanine,3,4-dichlorophenylalanine, 4-difluoromethylphenylalanine,2-methylphenylalanine, 2-naphthylalanine, tryptophan, 4-pyridinyl,4-bromophenylalanine, 3-pyridinyl, 4-trifluoromethylphenylalanine,4-carboxyphenylalanine, 4-methoxyphenylalanine, biphenylalanine, and3-chlorophenylalanine; and 2,4-diaminobutane.

In compounds of formula (I) where R² is not part of a ring, preferred R²side chains are: alanine, serine, and glycine.

In compounds of formula (I), preferred R³ side chains are: asparagine,aspartic acid, glutamic acid, glutamine, serine, ornithine, lysine,histidine, threonine, leucine, alanine, 2,3-diaminopropane, and2,4-diaminobutane.

In compounds of formula (I) where R⁴ is not part of a ring, preferred R⁴side chains are: valine, alanine, isoleucine, and glycine.

In compounds of formula (I), preferred R⁵ side chains are: aminomethane,histidine, asparagine, 2,3-diaminopropane, serine, glycine,2,4-diaminobutane, threonine, alanine, lysine, aspartic acid, alanine,and 3-thiazolylalanine.

In compounds of formula (I), preferred R⁶ side chains are: leucine,aspartic acid, asparagine, glutamic acid, glutamine, serine, lysine,3-cyclohexane, threonine, ornithine, 2,4-diaminobutane, alanine,arginine, and ornithine (COCH₃).

In compounds of formula (I) where R⁷ is not part of a ring, preferred R⁷side chains are: glycine, 2,4-diaminobutane, serine, lysine, arginine,ornithine, histidine, asparagine, glutamine, alanine, and2,4-diaminobutane (C(O)cyclobutane).

In compounds of formula (I) preferred R⁸ side chains are tryptophan and1,2-benzisothiazolinylalanine.

In compounds of formula (I) preferred R⁹ side chains are: serine,histidine, lysine, ornithine, 2,4-dibutylamine, threonine, lysine,glycine, glutamic acid, valine, 2,3-diaminopropane, arginine, asparticacid, and tyrosine.

In compounds of formula (I) preferred R¹⁰ side chains are: optionallysubstituted tryptophan, benzisothiazolylalanine, 1-napththylalanine, andmethionine.

In compounds of formula (I) preferred R¹¹ side chains are: norleucine,leucine, asparagine, phenylalanine, methionine, ethoxymethane, alanine,tryptophan, isoleucine, phenylpropane, glutamic acid, hexane, andheptane.

In compounds of formula (I) where R¹² is not part of a ring, preferredR¹² side chains are: norleucine, alanine, ethoxymethane, methionine,serine, phenylalanine, methoxyethane, leucine, tryptophan, isoleucine,glutamic acid, hexane, heptane, and glycine.

In compounds of formula (I) preferred R¹³ side chains: arginine,ornithine, alanine, 2,4-diaminobutane, 2,3-diaminopropane, leucine,aspartic acid, glutamic acid, serine, lysine, threonine,cyclopropylmethane, glycine, valine, isoleucine, histidine, and2-aminobutane.

In accordance with the present disclosure, we have discovered peptidesthat specifically bind to PD-L1 and are capable of inhibiting theinteraction of PD-L1 with PD-1 and CD80. These macrocyclic peptidesexhibit in vitro immunomodulatory efficacy thus making them therapeuticcandidates for the treatment of various diseases including cancer andinfectious diseases.

The terms “specific binding” or “specifically bind” refer to theinteraction between a protein and a binding molecule, such as a compoundor ligand. The interaction is dependent upon the presence of aparticular structure (i.e., an enzyme binding site, an antigenicdeterminant or epitope) of the protein that is recognized by the bindingmolecule. For example, if a compound has specific binding for proteinbinding site “A”, the presence of the compound in a reaction containinga protein including binding site A, and a labeled peptide thatspecifically binds to protein binding site A will reduce the amount oflabeled peptide bound to the protein. In contrast, nonspecific bindingof a compound to the protein does not result in aconcentration-dependent displacement of the labeled peptide from theprotein.

The present disclosure is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample and without limitation, isotopes of hydrogen include deuteriumand tritium. Isotopes of carbon include ¹³C and ¹⁴C.Isotopically-labeled compounds of the invention can generally beprepared by conventional techniques known to those skilled in the art orby processes analogous to those described herein, using an appropriateisotopically-labeled reagent in place of the non-labeled reagentotherwise employed. Such compounds may have a variety of potential uses,for example as standards and reagents in determining biologicalactivity. In the case of stable isotopes, such compounds may have thepotential to favorably modify biological, pharmacological, orpharmacokinetic properties.

An additional aspect of the subject matter described herein is the useof the disclosed peptides as radiolabeled ligands for development ofligand binding assays or for monitoring of in vivo adsorption,metabolism, distribution, receptor binding or occupancy, or compounddisposition. For example, a macrocyclic peptide described herein may beprepared using the radioactive isotope ¹²⁵I and the resultingradiolabeled peptide may be used to develop a binding assay or formetabolism studies. Alternatively, and for the same purpose, amacrocyclic peptide described herein may be converted to a radiolabeledform by catalytic tritiation using methods known to those skilled in theart.

The macrocyclic peptides of the present disclosure can also be used asPET imaging agents by adding a radioactive tracer using methods known tothose skilled in the art.

Preferred peptides include at least one of the macrocyclic peptidesprovided herein and these peptides may be included in pharmaceuticalcompositions and combinations.

The definitions provided herein apply, without limitation, to the termsas used throughout this specification, unless otherwise limited inspecific instances.

Those of ordinary skill in the art of amino acid and peptide chemistryare aware that an amino acid includes a compound represented by thegeneral structure:

-   -   where R and R′ are as discussed herein.

Unless otherwise indicated, the term “amino acid” as employed herein,alone or as part of another group, includes, without limitation, anamino group and a carboxyl group linked to the same carbon, referred toas “α” carbon, where R and/or R′ can be a natural or an un-natural sidechain, including hydrogen. The absolute “S” configuration at the “α”carbon is commonly referred to as the “L” or “natural” configuration. Inthe case where both the “R” and the “R′” (prime) substituents equalhydrogen, the amino acid is glycine and is not chiral.

The terms “natural amino acid side chain” and “naturally occurring aminoacid side chain,” as used herein, refer to side chain of any of thenaturally occurring amino acids (i.e., alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine) usually in theS-configuration (i.e., the L-amino acid).

The terms “unnatural amino acid side chain” and “non-naturally occurringamino acid side chain,” as used herein, refer to a side chain of anynaturally occurring amino acid usually in the R-configuration (i.e., theD-amino acid) or to a group other than a naturally occurring amino acidside chain in R- or S-configuration (i.e., the D- or L-amino acid,respectively) selected from:

-   -   C₂-C₇alkenyl, C₁-C₃alkoxyC₁-C₃alkyl,        C₁-C₆alkoxycarbonylC₁-C₃alkyl, C₁-C₇alkyl,        C₁-C₃alkylsulfanylC₁-C₃alkyl, amidoC₁-C₃alkyl, aminoC₁-C₃alkyl,        azaindolylC₁-C₃alkyl, benzothiazolylC₁-C₃alkyl,        benzothienylC₁-C₃alkyl, benzyloxyC₁-C₃alkyl, carboxyC₁-C₃alkyl,        C₃-C₁₄cycloalkylC₁-C₃alkyl, diphenylmethyl, furanylC₁-C₃alkyl,        imidazolylC₁-C₃alkyl, naphthylC₁-C₃alkyl, pyridinylC₁-C₃alkyl,        thiazolylC₁-C₃alkyl, thienylC₁-C₃alkyl;    -   biphenylC₁-C₃alkyl wherein the biphenyl is optionally        substituted with a methyl group;    -   heterorocyclyl optionally substituted with one, two, three,        four, or five groups independently selected from C₁-C₄alkoxy,        C₁-C₄alkyl, C₁-C₃alkylsulfonylamino, amido, amino,        aminoC₁-C₃alkyl, aminosulfonyl, carboxy, cyano, halo,        haloC₁-C₃alkyl, hydroxy, —NC(NH₂)₂, nitro, and —OP(O)(OH)₂;    -   indolylC₁-C₃alkyl, wherein the indolyl part is optionally        substituted with one group selected from C₁-C₃alkyl,        carboxyC₁-C₃alkyl, halo, hydroxy, and phenyl, wherein the phenyl        is further optionally substituted by one, two, or three groups        independently selected from C₁-C₃alkoxy, C₁-C₃alkyl, and halo;    -   NR^(y)R^(y′)(C₁-C₇alkyl), wherein R^(y) and R^(y′) are        independently selected from hydrogen, C₂-C₄alkenyloxycarbonyl,        C₁-C₃alkyl, C₁-C₃alkylcarbonyl, C₃-C₁₄cycloalkylcarbonyl,        furanylcarbonyl, and phenylcarbonyl. When the alkyl linker        contains more than one carbon an additional NR^(y)R^(y′) group        can be on the chain.

NR^(q)R^(t)carbonylC₁-C₃alkyl, wherein R^(q) and R^(t) are independentlyselected from hydrogen, C₁-C₃alkyl, and triphenylmethyl;

-   -   phenyl optionally substituted with one, two, three, four, or        five groups independently selected from C₁-C₄alkoxy, C₁-C₄alkyl,        C₁-C₃alkylsulfonylamino, amido, amino, aminoC₁-C₃alkyl,        aminosulfonyl, carboxy, cyano, halo, haloC₁-C₃alkyl, hydroxy,        —NC(NH₂)₂, nitro, and —OP(O)(OH)₂;    -   phenylC₁-C₃alkyl wherein the phenyl part is optionally        substituted with one, two, three, four, or five groups        independently selected from C₁-C₄alkoxy, C₁-C₄alkyl,        C₁-C₃alkylsulfonylamino, amido, amino, aminoC₁-C₃alkyl,        aminosulfonyl, carboxy, cyano, halo, haloC₁-C₃alkyl, hydroxy,        —NC(NH₂)₂, nitro, and —OP(O)(OH)₂; and    -   phenoxyC₁-C₃alkyl wherein the phenyl is optionally substituted        with a C₁-C₃alkyl group.

The term “C₂-C₄alkenyl,” as used herein, refers to a straight orbranched chain group of two to four carbon atoms containing at least onecarbon-carbon double bond.

The term “C₂-C₇alkenyl,” as used herein, refers to a straight orbranched chain group of two to seven carbon atoms containing at leastone carbon-carbon double bond.

The term “C₂-C₄alkenyloxy,” as used herein, refers to a C₂-C₄alkenylgroup attached to the parent molecular moiety through an oxygen atom.

The term “C₁-C₃alkoxy,” as used herein, refers to a C₁-C₃alkyl groupattached to the parent molecular moiety through an oxygen atom.

The term “C₁-C₄alkoxy,” as used herein, refers to a C₁-C₄alkyl groupattached to the parent molecular moiety through an oxygen atom.

The term “C₁-C₆alkoxy,” as used herein, refers to a C₁-C₆alkyl groupattached to the parent molecular moiety through an oxygen atom.

The term “C₁-C₃alkoxyC₁-C₃alkyl,” as used herein, refers to aC₁-C₃alkoxy group attached to the parent molecular moiety through aC₁-C₃alkyl group.

The term “C₁-C₆alkoxyC₁-C₆alkyl,” as used herein, refers to aC₁-C₆alkoxy group attached to the parent molecular moiety through aC₁-C₆alkyl group

The term “C₁-C₆alkoxycarbonyl,” as used herein, refers to a C₁-C₆alkoxygroup attached to the parent molecular moiety through a carbonyl group.

The term “C₁-C₆alkoxycarbonylC₁-C₃alkyl,” as used herein, refers to aC₁-C₆alkoxycarbonyl group attached to the parent molecular moietythrough a C₁-C₃alkyl group.

The term “C₁-C₃alkyl,” as used herein, refers to a group derived from astraight or branched chain saturated hydrocarbon containing from one tothree carbon atoms.

The term “C₁-C₄alkyl,” as used herein, refers to a group derived from astraight or branched chain saturated hydrocarbon containing from one tofour carbon atoms.

The term “C₁-C₆alkyl,” as used herein, refers to a group derived from astraight or branched chain saturated hydrocarbon containing from one tosix carbon atoms.

The term “C₁-C₃alkylcarbonyl,” as used herein, refers to a C₁-C₃alkylgroup attached to the parent molecular moiety through a carbonyl group.

The term “C₁-C₃alkylsulfanyl,” as used herein, refers to a C₁-C₃alkylgroup attached to the parent molecular moiety through a sulfur atom.

The term “C₁-C₃alkylsulfanylC₁-C₃alkyl,” as used herein, refers to aC₁-C₃alkylsulfanyl group attached to the parent molecular moiety througha C₁-C₃alkyl group.

The term “C₁-C₃alkylsulfonyl,” as used herein, refers to a C₁-C₃alkylgroup attached to the parent molecular moiety through a sulfonyl group.

The term “C₁-C₃alkylsulfonylamino,” as used herein, refers to aC₁-C₃alkylsulfonyl group attached to the parent molecular moiety throughan amino group.

The term “amido,” as used herein, refers to —C(O)NH₂.

The term “amidoC₁-C₃alkyl,” as used herein, refers to an amido groupattached to the parent molecular moiety through a C₁-C₃alkyl group.

The term “amino,” as used herein, refers to —NH₂.

The term “aminoC₁-C₃alkyl,” as used herein, refers to an amino groupattached to the parent molecular moiety through a C₁-C₃alkyl group.

The term “aminosulfonyl,” as used herein, refers to an amino groupattached to the parent molecular moiety through a sulfonyl group.

The term “azaindolylC₁-C₃alkyl,” as used herein, refers to an azaindolylgroup attached to the parent molecular through a C₁-C₃alkyl group. Theazaindolyl group can be attached to the alkyl moiety through anysubstitutable atom in the group.

The term “benzothiazolylC₁-C₃alkyl,” as used herein, refers to anbenzothiazolyl group attached to the parent molecular through aC₁-C₃alkyl group. The benzothiazolyl group can be attached to the alkylmoiety through any substitutable atom in the group.

The term “benzothienylC₁-C₃alkyl,” as used herein, refers to abenzothienyl group attached to the parent molecular through a C₁-C₃alkylgroup. The benzothienyl group can be attached to the alkyl moietythrough any substitutable atom in the group.

The term “benzyloxy,” as used herein, refers to a benzyl group attachedto the parent molecular moiety through an oxygen atom.

The term “benzyloxyC₁-C₃alkyl,” as used herein, refers to a benzyloxygroup attached to the parent molecular moiety through a C₁-C₃alkylgroup.

The term “biphenylC₁-C₃alkyl,” as used herein, refers to a biphenylgroup attached to the parent molecular moiety through a C₁-C₃alkylgroup. The biphenyl group can be attached to the alkyl moiety throughany substitutable atom in the group.

The term “carbonyl,” as used herein, refers to —C(O)—.

The term “carboxy,” as used herein, refers to —CO₂H.

The term “carboxyC₁-C₃alkyl,” as used herein, refers to a carboxy groupattached to the parent molecular moiety through a C₁-C₃alkyl group.

The term “carboxyC₁-C₆alkyl,” as used herein, refers to a carboxy groupattached to the parent molecular moiety through a C₁-C₆alkyl group.

The term “cyano,” as used herein, refers to —CN.

The term “C₃-C₁₄cycloalkyl,” as used herein, refers to a saturatedmonocyclic, bicyclic, or tricyclic hydrocarbon ring system having threeto fourteen carbon atoms and zero heteroatoms. The bicyclic andtricyclic rings may be fused, spirocyclic, or bridged. Representativeexamples of cycloalkyl groups include, but are not limited to,cyclopropyl, cyclopentyl, bicyclo[3.1.1]heptyl, and adamantyl.

The term “C₃-C₁₄cycloalkylC₁-C₃alkyl,” as used herein, refers to aC₃-C₁₄cycloalkyl group attached to the parent molecular moiety through aC₁-C₃alkyl group.

The term “C₃-C₁₄cycloalkylcarbonyl,” as used herein, refers to a C₃-C₁₄cycloalkyl group attached to the parent molecular moiety through acarbonyl group.

The term “furanylC₁-C₃alkyl,” as used herein, refers to a furanyl groupattached to the parent molecular moiety through a C₁-C₃alkyl group. Thefuranyl group can be attached to the alkyl moiety through anysubstitutable atom in the group.

The term “furanylcarbonyl,” as used herein, refers to a furanyl groupattached to the parent molecular moiety through a carbonyl group.

The terms “halo” and “halogen,” as used herein, refer to F, Cl, Br, orI.

The term “haloC₁-C₃alkyl,” as used herein, refers to a C₁-C₃alkyl groupsubstituted with one, two, or three halogen atoms.

The term “haloC₁-C₆alkyl,” as used herein, refers to a C₁-C₆alkyl groupsubstituted with one, two, or three halogen atoms.

The term “halomethyl,” as used herein, refers to a methyl groupsubstituted with one, two, or three halogen atoms.

The term “heterocyclyl,” as used herein, refers to a five-, six-, orseven-membered ring containing one, two, or three heteroatomsindependently selected from nitrogen, oxygen, and sulfur. Thefive-membered ring has zero to two double bonds and the six- andseven-membered rings have zero to three double bonds. The term“heterocyclyl” also includes bicyclic groups in which the heterocyclylring is fused to a four- to six-membered aromatic or non-aromaticcarbocyclic ring or another monocyclic heterocyclyl group. Theheterocyclyl groups of the present disclosure are attached to the parentmolecular moiety through a carbon atom in the group. Examples ofheterocyclyl groups include, but are not limited to, benzothienyl,furyl, imidazolyl, indolinyl, indolyl, isothiazolyl, isoxazolyl,morpholinyl, oxazolyl, piperazinyl, piperidinyl, pyrazolyl, pyridinyl,pyrrolidinyl, pyrrolopyridinyl, pyrrolyl, thiazolyl, thienyl, andthiomorpholinyl.

The term “hydroxy,” as used herein, refers to —OH.

The term “hydroxyC₁-C₆alkyl,” as used herein, refers to a C₁-C₆alkylgroup substituted with one, two, or three hydroxy groups.

The term “imidazolylC₁-C₃alkyl,” as used herein, refers to an imidazolylgroup attached to the parent molecular moiety through a C₁-C₃alkylgroup. The imidazolyl group can be attached to the alkyl moiety throughany substitutable atom in the group.

The term “indolylC₁-C₃alkyl,” as used herein, refers to an indolyl groupattached to the parent molecular moiety through a C₁-C₃alkyl group. Theindolyl group can be attached to the alkyl moiety through anysubstitutable atom in the group.

The term “naphthylC₁-C₃alkyl,” as used herein, refers to a naphthylgroup attached to the parent molecular moiety through a C₁-C₃alkylgroup. The naphthyl group can be attached to the alkyl moiety throughany substitutable atom in the group.

The term “nitro,” as used herein, refers to —NO₂.

The term “NR^(y)R^(y′),” as used herein, refers to two groups, R^(y) andR^(y′), which are attached to the parent molecular moiety through anitrogen atom. R^(y) and R^(y′) are independently selected fromhydrogen, C₂-C₄alkenyloxycarbonyl, C₁-C₃alkylcarbonyl,C₃-C₁₄cycloalkylcarbonyl, furanylcarbonyl, and phenylcarbonyl.

The term “NR^(y)R^(y′)(C₁-C₃)alkyl,” as used herein, refers to anNR^(y)R^(y′) group attached to the parent molecular moiety through aC₁-C₃alkyl group.

The term “NR^(y)R^(y′),” as used herein, refers to two groups, R^(q) andR^(t), which are attached to the parent molecular moiety through anitrogen atom. R^(q) and R^(t) are independently selected from hydrogen,C₁-C₃alkyl, and triphenylmethyl.

The term “NR^(q)R^(t)carbonyl,” as used herein, refers to an NR^(q)R^(t)group attached to the parent molecular moiety through a carbonyl group.

The term “NR^(q)R^(t) carbonylC₁-C₃alkyl,” as used herein, refers to anNR^(q)R^(t)carbonyl group attached to the parent molecular moietythrough a C₁-C₃alkyl group.

The tem “phenoxy,” as used herein, refers to a phenyl group attached tothe parent molecular moiety through an oxygen atom.

The term “phenoxyC₁-C₃alkyl,” as used herein, refers to a phenoxy groupattached to the parent molecular moiety through a C₁-C₃alkyl group.

The term “phenylC₁-C₃alkyl,” as used herein, refers to a phenyl groupattached to the parent molecular moiety through a C₁-C₃alkyl group.

The term “phenylC₁-C₆alkyl,” as used herein, refers to a phenyl groupattached to the parent molecular moiety through a C₁-C₆alkyl group.

The term “phenylcarbonyl,” as used herein, refers to a phenyl groupattached to the parent molecular moiety through a carbonyl group.

The term “pyridinylC₁-C₃alkyl,” as used herein, refers to a pyridinylgroup attached to the parent molecular moiety through a C₁-C₃alkylgroup. The pyridinyl group can be attached to the alkyl moiety throughany substitutable atom in the group.

The term “sulfanyl,” as used herein, refers to —S—.

The term “sulfonyl,” as used herein, refers to —SO₂—.

The term “thiazolylC₁-C₃alkyl,” as used herein, refers to a thiazolylgroup attached to the parent molecular moiety through a C₁-C₃alkylgroup. The thiazolyl group can be attached to the alkyl moiety throughany substitutable atom in the group.

The term “thienylC₁-C₃alkyl,” as used herein, refers to a thienyl groupattached to the parent molecular moiety through a C₁-C₃alkyl group. Thethienyl group can be attached to the alkyl moiety through anysubstitutable atom in the group.

The term “treating” refers to: (i) preventing a disease, disorder, orcondition from occurring in a patient that may be predisposed to thedisease, disorder, and/or condition but has not yet been diagnosed ashaving it; (ii) inhibiting the disease, disorder, or condition, i.e.,arresting its development; and (iii) relieving the disease, disorder, orcondition, i.e., causing regression of the disease, disorder, and/orcondition and/or symptoms associated with the disease, disorder, and/orcondition.

Binding of the macrocyclic peptides to PD-L1 can be measured, forexample, by methods such as homogeneous time-resolved fluorescence(HTRF), Surface Plasmon Resonance (SPR), isothermal titrationcalorimetry (ITC), nuclear magnetic resonance spectroscopy (NMR), andthe like. Further, binding of the macrocyclic peptides to PD-L1expressed on the surface of cells can be measured as described herein incellular binding assays.

Administration of a therapeutic agent described herein includes, withoutlimitation, administration of a therapeutically effective amount oftherapeutic agent. The term “therapeutically effective amount” as usedherein refers, without limitation, to an amount of a therapeutic agentto treat or prevent a condition treatable by administration of acomposition of the PD-1/PD-L1 binding inhibitors described herein. Thatamount is the amount sufficient to exhibit a detectable therapeutic orpreventative or ameliorative effect. The effect may include, for exampleand without limitation, treatment or prevention of the conditions listedherein. The precise effective amount for a subject will depend upon thesubject's size and health, the nature and extent of the condition beingtreated, recommendations of the treating physician, and therapeutics orcombination of therapeutics selected for administration. Thus, it is notuseful to specify an exact effective amount in advance.

In another aspect, the disclosure pertains to methods of inhibitinggrowth of tumor cells in a subject using the macrocyclic peptides of thepresent disclosure. As demonstrated herein, the macrocyclic peptides ofthe present disclosure are capable of binding to PD-L1, disrupting theinteraction between PD-L1 and PD-1, competing with the binding of PD-L1with anti-PD-1 monoclonal antibodies that are known to block theinteraction with PD-1, enhancing CMV-specific T cell IFNγ secretion, andenhancement of HIV-specific T cell IFNg secretion. As a result, themacrocyclic peptides of the present disclosure are useful for modifyingan immune response, treating diseases such as cancer or infectiousdisease, stimulating a protective autoimmune response or to stimulateantigen-specific immune responses (e.g., by coadministration of PD-L1blocking peptides with an antigen of interest).

In order that the present disclosure may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The terms “Programmed Death Ligand 1”, “Programmed Cell Death Ligand 1”,“Protein PD-L1”, “PD-L1”, “PDL1”, “PDCDL1”, “hPD-L1”, “hPD-LI”, “CD274”and “B7-H1” are used interchangeably, and include variants, isoforms,species homologs of human PD-L1, and analogs having at least one commonepitope with PD-L1. The complete PD-L1 sequence can be found underGENBANK® Accession No. NP_054862.

The terms “Programmed Death 1”, “Programmed Cell Death 1”, “ProteinPD-1”, “PD-1”, “PD1”, “PDCD1”, “hPD-1” and “hPD-1” are usedinterchangeably, and include variants, isoforms, species homologs ofhuman PD-1, and analogs having at least one common epitope with PD-1.The complete PD-1 sequence can be found under GENBANK® Accession No.U64863.

The terms “cytotoxic T lymphocyte-associated antigen-4”, “CTLA-4”,“CTLA4”, “CTLA-4 antigen” and “CD152” (see, e.g., Murata, Am. J.Pathol., 155:453-460 (1999)) are used interchangeably, and includevariants, isoforms, species homologs of human CTLA-4, and analogs havingat least one common epitope with CTLA-4 (see, e.g., Balzano, Int. J.Cancer Suppl., 7:28-32 (1992)). The complete CTLA-4 nucleic acidsequence can be found under GENBANK® Accession No. L15006.

The term “immune response” refers to the action of, for example,lymphocytes, antigen presenting cells, phagocytic cells, granulocytes,and soluble macromolecules produced by the above cells or the liver(including macrocyclic peptides, cytokines, and complement) that resultsin selective damage to, destruction of, or elimination from the humanbody of invading pathogens, cells or tissues infected with pathogens,cancerous cells, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues.

An “adverse event” (AE) as used herein is any unfavorable and generallyunintended, even undesirable, sign (including an abnormal laboratoryfinding), symptom, or disease associated with the use of a medicaltreatment. For example, an adverse event may be associated withactivation of the immune system or expansion of immune system cells(e.g., T cells) in response to a treatment. A medical treatment may haveone or more associated AEs and each AE may have the same or differentlevel of severity. Reference to methods capable of “altering adverseevents” means a treatment regime that decreases the incidence and/orseverity of one or more AEs associated with the use of a differenttreatment regime.

As used herein, “hyperproliferative disease” refers to conditionswherein cell growth is increased over normal levels. For example,hyperproliferative diseases or disorders include malignant diseases(e.g., esophageal cancer, colon cancer, biliary cancer) andnon-malignant diseases (e.g., atherosclerosis, benign hyperplasia, andbenign prostatic hypertrophy).

As used herein, “about” or “comprising essentially of” mean within anacceptable error range for the particular value as determined by one ofordinary skill in the art, which will depend in part on how the value ismeasured or determined, i.e., the limitations of the measurement system.For example, “about” or “comprising essentially of” can mean within oneor more than one standard deviation per the practice in the art.Alternatively, “about” or “comprising essentially of” can mean a rangeof up to 20%. Furthermore, particularly with respect to biologicalsystems or processes, the terms can mean up to an order of magnitude orup to 5-fold of a value. When particular values are provided in theapplication and claims, unless otherwise stated, the meaning of “about”or “comprising essentially of” should be assumed to be within anacceptable error range for that particular value.

As described herein, any concentration range, percentage range, ratiorange or integer range is to be understood to include the value of anyinteger within the recited range and, when appropriate, fractionsthereof (such as one tenth and one hundredth of an integer), unlessotherwise indicated.

Competition Assays

The present disclosure is also directed to macrocyclic peptides that arecapable of competing with the binding of a reference anti-PD-L1 antibody(MDX-1105) by at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, and at least about 100%. Suchmacrocyclic peptides may share structural homology with one or moremacrocyclic peptides disclosed herein, including mutant, conservativesubstitution, functional substitution, and deletion forms, provided theyspecific bind to PD-L1. For example, if a macrocyclic peptide bindssubstantially to the same region of PD-L1 as a reference anti-PD-L1antibody, the macrocyclic peptide should bind to an epitope of PD-L1that at least overlaps with the PD-L1 epitope that the anti-PD-L1monoclonal antibody binds to. The overlapping region can range from oneamino acid residue to several hundred amino acid residues. Themacrocyclic peptide should then compete with and/or block the binding ofthe anti-PD-L1 monoclonal antibody to PD-L1 and thereby decrease thebinding of the anti-PD-L1 monoclonal antibody to PD-L1, preferably by atleast about 50% in a competition assay.

Anti-PD-L1 antibodies that may be used as reference antibodies forcompetition assay purposes are known in the art. For example, thefollowing representative anti-PD-L1 antibodies may be used: MDX-1105(BMS); L01X-C (Serono), L1X3 (Serono), MSB-0010718C (Serono), and PD-L1Probody (CytomX), and the PD-L1 antibodies disclosed in co-owned WO2007/005874.

Anti-PD-1 antibodies that may be used as reference antibodies forcompetition assay purposes are known in the art. For example, thefollowing representative anti-PD-1 antibodies may be used: nivolumab(BMS); 17D8, 2D3, 4H1, 4A11, 7D3 and 5F4 each disclosed in co-owned U.S.Pat. No. 8,008,449 (BMS), MK-3475 (Merck, disclosed in U.S. Pat. No.8,168,757), and the antibodies disclosed in U.S. Pat. No. 7,488,802.

Pharmaceutical Compositions

In another aspect, the present disclosure provides a composition, e.g.,a pharmaceutical composition, containing one or a combination ofmacrocyclic peptides of the present disclosure, formulated together witha pharmaceutically acceptable carrier. Such compositions may include oneor a combination of (e.g., two or more different) macrocyclic peptides,or immunoconjugates or bispecific molecules of the disclosure. Forexample, a pharmaceutical composition of the disclosure can comprise acombination of macrocyclic peptides (or immunoconjugates or bispecifics)that bind to different epitopes on the target antigen or that havecomplementary activities.

Pharmaceutical compositions of the disclosure also can be administeredin combination therapy, i.e., combined with other agents. For example,the combination therapy can include a macrocyclic peptide combined withat least one other anti-inflammatory or immunosuppressant agent.Examples of therapeutic agents that can be used in combination therapyare described in greater detail below in the section on uses of themacrocyclic peptides of the disclosure.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., a macrocyclicpeptide, immunoconjugate, or bispecific molecule, may be coated in amaterial to protect the compound from the action of acids and othernatural conditions that may inactivate the compound.

The pharmaceutical compounds of the disclosure may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”or “therapeutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound and does not impartany undesired toxicological effects (see e.g., Berge, S. M. et al., J.Pharm. Sci., 66:1-19 (1977)). Examples of such salts include acidaddition salts and base addition salts. Acid addition salts includethose derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous andthe like, as well as from nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acidsand the like. Base addition salts include those derived from alkalineearth metals, such as sodium, potassium, magnesium, calcium and thelike, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the disclosure also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe disclosure is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, preferably from about0.1 percent to about 70 percent, most preferably from about 1 percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of therapeutic situation. It is especially advantageous toformulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the disclosure are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

For administration of the macrocyclic peptide, the dosage ranges fromabout 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the hostbody weight. For example dosages can be 0.3 mg/kg body weight, 1 mg/kgbody weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg bodyweight or within the range of 1-10 mg/kg. An exemplary treatment regimeentails administration once per day, twice per day, bi-weekly,tri-weekly, weekly, once every two weeks, once every three weeks, onceevery four weeks, once a month, once every 3 months or once every threeto 6 months. Preferred dosage regimens for a macrocyclic peptide of thedisclosure include 1 mg/kg body weight or 3 mg/kg body weight viaintravenous administration, with the macrocycle being given using one ofthe following dosing schedules: (i) every four weeks for six dosages,then every three months; (ii) every three weeks; (iii) 3 mg/kg bodyweight once followed by 1 mg/kg body weight every three weeks.

In some methods, two or more macrocyclic peptides with different bindingspecificities are administered simultaneously, in which case the dosageof each compound administered falls within the ranges indicated. Thecompounds are usually administered on multiple occasions. Intervalsbetween single dosages can be, for example, weekly, monthly, every threemonths or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of macrocyclic peptide to the target antigen inthe patient. In some methods, dosage is adjusted to achieve a plasmaconcentration of about 1-1000 .mu.g/ml and in some methods about 25-300.mu.g/ml.

Alternatively, the macrocyclic peptide can be administered as asustained release formulation, in which case less frequentadministration is required. The dosage and frequency of administrationcan vary depending on whether the treatment is prophylactic ortherapeutic. In prophylactic applications, a relatively low dosage isadministered at relatively infrequent intervals over a long period oftime. Some patients continue to receive treatment for the rest of theirlives. In therapeutic applications, a relatively high dosage atrelatively short intervals is sometimes required until progression ofthe disease is reduced or terminated, and preferably until the patientshows partial or complete amelioration of symptoms of disease.Thereafter, the patient can be administered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present disclosure may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentdisclosure employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A “therapeutically effective dosage” of a macrocyclic peptide of thedisclosure preferably results in a decrease in severity of diseasesymptoms, an increase in frequency and duration of disease symptom-freeperiods, or a prevention of impairment or disability due to the diseaseaffliction. For example, for the treatment of tumors, a “therapeuticallyeffective dosage” preferably inhibits cell growth or tumor growth by atleast about 20%, more preferably by at least about 40%, even morepreferably by at least about 60%, and still more preferably by at leastabout 80% relative to untreated subjects. The ability of a compound toinhibit tumor growth and/or HIV can be evaluated in an animal modelsystem predictive of efficacy in human tumors or viral efficacy.Alternatively, this property of a composition can be evaluated byexamining the ability of the compound to inhibit, such inhibition invitro by assays known to the skilled practitioner. A therapeuticallyeffective amount of a therapeutic compound can decrease tumor size,decrease viral load, or otherwise ameliorate symptoms in a subject. Oneof ordinary skill in the art would be able to determine such amountsbased on such factors as the subject's size, the severity of thesubject's symptoms, and the particular composition or route ofadministration selected.

In another aspect, the instant disclosure provides a pharmaceutical kitof parts comprising a macrocyclic peptide and an another immumodulator,as described herein. The kit may also further comprise instructions foruse in the treatment of a hyperproliferative disease (such as cancer asdescribed herein) and/or anti-viral disease.

A composition of the present disclosure can be administered via one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Preferred routes of administration for macrocyclic peptides ofthe disclosure include intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, spinal or other parenteral routes ofadministration, for example by injection or infusion. The phrase“parenteral administration” as used herein means modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion.

Alternatively, a macrocyclic peptide of the disclosure can beadministered via a non-parenteral route, such as a topical, epidermal ormucosal route of administration, for example, intranasally, orally,vaginally, rectally, sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Robinson, J. R.,ed., Sustained and Controlled Release Drug Delivery Systems, MarcelDekker, Inc., New York (1978).

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of the disclosure can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824,or 4,596,556. Examples of well-known implants and modules useful in thepresent disclosure include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medication through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Thesepatents are incorporated herein by reference. Many other such implants,delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the macrocyclic peptides of the disclosure canbe formulated to ensure proper distribution in vivo. For example, theblood-brain barrier (BBB) excludes many highly hydrophilic compounds. Toensure that therapeutic compounds of the disclosure cross the BBB (ifdesired), they can be formulated, for example, in liposomes. For methodsof manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811,5,374,548, and 5,399,331. The liposomes may comprise one or moremoieties which are selectively transported into specific cells ororgans, thus enhance targeted drug delivery (see, e.g., Ranade, V. V.,J. Clin. Pharmacol., 29:685 (1989)). Exemplary targeting moietiesinclude folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low etal.); mannosides (Umezawa et al., Biochem. Biophys. Res. Commun.,153:1038 (1988)); macrocyclic peptides (Bloeman, P. G. et al., FEBSLett., 357:140 (1995); Owais, M. et al., Antimicrob. Agents Chemother.,39:180 (1995)); surfactant protein A receptor (Briscoe et al., Am. J.Physiol., 1233:134 (1995)); p 120 (Schreier et al., J. Biol. Chem.,269:9090 (1994)); see also Keinanen, K. et al., FEBS Lett., 346:123(1994); Killion, J. J. et al., Immunomethods 4:273 (1994).

Uses and Methods of the Disclosure

The macrocyclic peptides, compositions and methods of the presentdisclosure have numerous in vitro and in vivo utilities involving, forexample, detection of PD-L1 or enhancement of immune response byblockade of PD-L1. For example, these molecules can be administered tocells in culture, in vitro or ex vivo, or to human subjects, e.g., invivo, to enhance immunity in a variety of situations. Accordingly, inone aspect, the disclosure provides a method of modifying an immuneresponse in a subject comprising administering to the subject themacrocyclic peptide of the disclosure such that the immune response inthe subject is modified. Preferably, the response is enhanced,stimulated or up-regulated. In other respects, the macrocyclic peptidemay have anti-cyno, anti-mouse, and/or anti-woodchuck binding andtherapeutic activity.

As used herein, the term “subject” is intended to include human andnon-human animals. Non-human animals includes all vertebrates, e.g.,mammals and non-mammals, such as non-human primates, sheep, dogs, cats,cows, horses, chickens, woodchuck, amphibians, and reptiles, althoughmammals are preferred, such as non-human primates, sheep, dogs, cats,cows and horses. Preferred subjects include human patients in need ofenhancement of an immune response. The methods are particularly suitablefor treating human patients having a disorder that can be treated byaugmenting the T-cell mediated immune response. In a particularembodiment, the methods are particularly suitable for treatment ofcancer cells in vivo. To achieve antigen-specific enhancement ofimmunity, the macrocyclic peptides can be administered together with anantigen of interest. When macrocyclic peptides to PD-L1 are administeredtogether with another agent, the two can be administered in either orderor simultaneously.

The disclosure further provides methods for detecting the presence ofhuman, woodchuck, cyno, and/or mouse PD-L1 antigen in a sample, ormeasuring the amount of human, woodchuck, cyno, and/or mouse PD-L1antigen, comprising contacting the sample, and a control sample, with areference macrocyclic peptide which specifically binds to human,woodchuck, cyno, and/or mouse PD-L1, under conditions that allow forformation of a complex between the macrocycle and human, woodchuck,cyno, and/or mouse PD-L1. The formation of a complex is then detected,wherein a difference complex formation between the sample compared tothe control sample is indicative the presence of human, woodchuck, cyno,and/or mouse PD-L1 antigen in the sample.

Given the specific binding of the macrocyclic peptides of the disclosurefor PD-L1, compared to CD28, ICOS and CTLA-4, the macrocyclic peptidesof the disclosure can be used to specifically detect PD-L1 expression onthe surface of cells and, moreover, can be used to purify PD-L1 viaimmunoaffinity purification.

Cancer

Blockade of PD-1 by macrocyclic peptides can enhance the immune responseto cancerous cells in the patient. The ligand for PD-1, PD-L1, is notexpressed in normal human cells, but is abundant in a variety of humancancers (Dong et al., Nat. Med., 8:787-789 (2002)). The interactionbetween PD-1 and PD-L1 results in a decrease in tumor infiltratinglymphocytes, a decrease in T-cell receptor mediated proliferation, andimmune evasion by the cancerous cells (Dong et al., J. Mol. Med.,81:281-287 (2003); Blank et al., Cancer Immunol. Immunother., 54:307-314(2005); Konishi et al., Clin. Cancer Res., 10:5094-5100 (2004)). Immunesuppression can be reversed by inhibiting the local interaction of PD-1to PD-L1 and the effect is additive when the interaction of PD-1 toPD-L2 is blocked as well (Iwai et al., Proc. Natl. Acad. Sci.,99:12293-12297 (2002); Brown et al., J. Immunol., 170:1257-1266 (2003)).While previous studies have shown that T-cell proliferation can berestored by inhibiting the interaction of PD-1 to PD-L1, there have beenno reports of a direct effect on cancer tumor growth in vivo by blockingthe PD-1/PD-L1 interaction. In one aspect, the present disclosurerelates to treatment of a subject in vivo using a macrocyclic peptidesuch that growth of cancerous tumors is inhibited. A macrocyclic peptidemay be used alone to inhibit the growth of cancerous tumors.Alternatively, a macrocyclic peptide may be used in conjunction withother immunogenic agents, standard cancer treatments, or othermacrocyclic peptides, as described below.

Accordingly, in one embodiment, the disclosure provides a method ofinhibiting growth of tumor cells in a subject, comprising administeringto the subject a therapeutically effective amount of a macrocyclicpeptide.

Preferred cancers whose growth may be inhibited using the macrocyclicpeptides of the disclosure include cancers typically responsive toimmunotherapy. Non-limiting examples of preferred cancers for treatmentinclude melanoma (e.g., metastatic malignant melanoma), renal cellcarcinoma (e.g., clear cell carcinoma), prostate cancer (e.g., hormonerefractory prostate adenocarcinoma and castration-resistant prostatecancer), breast cancer, colorectal cancer and lung cancer (e.g.,squamous and non-squamous non-small cell lung cancer). Additionally, thedisclosure includes refractory or recurrent malignancies whose growthmay be inhibited using the macrocyclic peptides of the disclosure.

Examples of other cancers that may be treated using the methods of thedisclosure include bone cancer, pancreatic cancer, skin cancer, cancerof the head or neck, cutaneous or intraocular malignant melanoma,uterine cancer, ovarian cancer, colon cancer, rectal cancer, cancer ofthe anal region, stomach/gastric cancer, testicular cancer, uterinecancer, carcinoma of the fallopian tubes, carcinoma of the endometrium,carcinoma of the cervix, carcinoma of the vagina, carcinoma of thevulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, chronic or acute leukemias including acutemyeloid leukemia, chronic myeloid leukemia, acute lymphoblasticleukemia, chronic lymphocytic leukemia, solid tumors of childhood,lymphocytic lymphoma, cancer of the bladder, cancer of the kidney orureter, carcinoma of the renal pelvis, neoplasm of the central nervoussystem (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axistumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally induced cancers including those induced by asbestos, andcombinations of said cancers. The present disclosure is also useful fortreatment of metastatic cancers, especially metastatic cancers thatexpress PD-L1 (Iwai et al., Int. Immunol., 17: 133-144 (2005)).

Optionally, macrocyclic peptides to PD-L1 can be combined with animmunogenic agent, such as cancerous cells, purified tumor antigens(including recombinant proteins, peptides, and carbohydrate molecules),cells, and cells transfected with genes encoding immune stimulatingcytokines (He et al., J. Immunol., 173:4919-4928 (2004)). Non-limitingexamples of tumor vaccines that can be used include peptides of melanomaantigens, such as peptides of gp100, MAGE antigens, Trp-2, MART1 and/ortyrosinase, or tumor cells transfected to express the cytokine GM-CSF(discussed further below).

In humans, some tumors have been shown to be immunogenic such asmelanomas. It is anticipated that by raising the threshold of T cellactivation by PD-L1 blockade, we may expect to activate tumor responsesin the host.

PD-L1 blockade is likely to be most effective when combined with avaccination protocol. Many experimental strategies for vaccinationagainst tumors have been devised (see Rosenberg, S., Development ofCancer Vaccines, ASCO Educational Book Spring: 60-62 (2000); Logothetis,C., ASCO Educational Book Spring: 300-302 (2000); Khayat, D., ASCOEducational Book Spring: 414-428 (2000); Foon, K., ASCO Educational BookSpring: 730-738 (2000); see also Restifo, N. et al., Cancer Vaccines,Chapter 61, pp. 3023-3043, in DeVita, V. et al., eds., Cancer:Principles and Practice of Oncology, Fifth Edition (1997)). In one ofthese strategies, a vaccine is prepared using autologous or allogeneictumor cells. These cellular vaccines have been shown to be mosteffective when the tumor cells are transduced to express GM-CSF. GM-CSFhas been shown to be a potent activator of antigen presentation fortumor vaccination (Dranoff et al., Proc. Natl. Acad. Sci. USA, 90:3539-3543 (1993)).

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so called tumor specificantigens (Rosenberg, S. A., Immunity, 10:281-287 (1999)). In many cases,these tumor specific antigens are differentiated antigens expressed inthe tumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens, and Trp-2. More importantly,many of these antigens can be shown to be the targets of tumor specificT cells found in the host. PD-L1 blockade may be used in conjunctionwith a collection of recombinant proteins and/or peptides expressed in atumor in order to generate an immune response to these proteins. Theseproteins are normally viewed by the immune system as self antigens andare therefore tolerant to them. The tumor antigen may also include theprotein telomerase, which is required for the synthesis of telomeres ofchromosomes and which is expressed in more than 85% of human cancers andin only a limited number of somatic tissues (Kim, N et al., Science,266:2011-2013 (1994)). (These somatic tissues may be protected fromimmune attack by various means). Tumor antigen may also be“neo-antigens” expressed in cancer cells because of somatic mutationsthat alter protein sequence or create fusion proteins between twounrelated sequences (i.e., bcr-abl in the Philadelphia chromosome), oridiotype from B cell tumors.

Other tumor vaccines may include the proteins from viruses implicated inhuman cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses(HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). Another form oftumor specific antigen which may be used in conjunction with PD-L1blockade is purified heat shock proteins (HSP) isolated from the tumortissue itself. These heat shock proteins contain fragments of proteinsfrom the tumor cells and these HSPs are highly efficient at delivery toantigen presenting cells for eliciting tumor immunity (Suot, R. et al.,Science, 269:1585-1588 (1995); Tamura, Y. et al., Science, 278:117-120(1997)).

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DC's can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle, F. et al., Nat. Med., 4:328-332 (1998)). DCs mayalso be transduced by genetic means to express these tumor antigens aswell. DCs have also been fused directly to tumor cells for the purposesof immunization (Kugler, A. et al., Nat. Med., 6:332-336 (2000)). As amethod of vaccination, DC immunization may be effectively combined withPD-L1 blockade to activate more potent anti-tumor responses.

PD-L1 blockade may also be combined with standard cancer treatments.PD-L1 blockade may be effectively combined with chemotherapeuticregimes. In these instances, it may be possible to reduce the dose ofchemotherapeutic reagent administered (Mokyr, M. et al., Cancer Res.,58:5301-5304 (1998)). An example of such a combination is a macrocyclicpeptide in combination with decarbazine for the treatment of melanoma.Another example of such a combination is a macrocyclic peptide incombination with interleukin-2 (IL-2) for the treatment of melanoma. Thescientific rationale behind the combined use of PD-L1 blockade andchemotherapy is that cell death, that is a consequence of the cytotoxicaction of most chemotherapeutic compounds, should result in increasedlevels of tumor antigen in the antigen presentation pathway. Othercombination therapies that may result in synergy with PD-L1 blockadethrough cell death are radiation, surgery, and hormone deprivation. Eachof these protocols creates a source of tumor antigen in the host.Angiogenesis inhibitors may also be combined with PD-L1 blockade.Inhibition of angiogenesis leads to tumor cell death which may feedtumor antigen into host antigen presentation pathways.

PD-L1 blocking macrocyclic peptides can also be used in combination withbispecific macrocyclic peptides that target Fc alpha or Fc gammareceptor-expressing effectors cells to tumor cells (see, e.g., U.S. Pat.Nos. 5,922,845 and 5,837,243). Bispecific macrocyclic peptides can beused to target two separate antigens. For example anti-Fc receptor/antitumor antigen (e.g., Her-2/neu) bispecific macrocyclic peptides havebeen used to target macrophages to sites of tumor. This targeting maymore effectively activate tumor specific responses. The T cell arm ofthese responses would be augmented by the use of PD-L1 blockade.Alternatively, antigen may be delivered directly to DCs by the use ofbispecific macrocyclic peptides which bind to tumor antigen and adendritic cell specific cell surface marker.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation of proteinswhich are expressed by the tumors and which are immunosuppressive. Theseinclude among others TGF-beta (Kehrl, J. et al., J. Exp. Med.,163:1037-1050 (1986)), IL-10 (Howard, M. et al., Immunology Today,13:198-200 (1992)), and Fas ligand (Hahne, M. et al., Science,274:1363-1365 (1996)). Macrocyclic peptides to each of these entitiesmay be used in combination with anti-PD-L1 to counteract the effects ofthe immunosuppressive agent and favor tumor immune responses by thehost.

Other macrocyclic peptides which may be used to activate host immuneresponsiveness can be used in combination with anti-PD-L1. These includemolecules on the surface of dendritic cells which activate DC functionand antigen presentation. Anti-CD40 macrocyclic peptides are able tosubstitute effectively for T cell helper activity (Ridge, J. et al.,Nature, 393:474-478 (1998)) and can be used in conjunction with PD-1antibodies (Ito, N. et al., Immunobiology, 201(5):527-540 (2000)).Activating macrocyclic peptides to T cell costimulatory molecules suchas CTLA-4 (e.g., U.S. Pat. No. 5,811,097), OX-40 (Weinberg, A. et al.,Immunol., 164:2160-2169 (2000)), 4-1BB (Melero, I. et al., Nat. Med.,3:682-685 (1997), and ICOS (Hutloff, A. et al., Nature, 397:262-266(1999)) may also provide for increased levels of T cell activation.

Bone marrow transplantation is currently being used to treat a varietyof tumors of hematopoietic origin. While graft versus host disease is aconsequence of this treatment, therapeutic benefit may be obtained fromgraft vs. tumor responses. PD-L1 blockade can be used to increase theeffectiveness of the donor engrafted tumor specific T cells.

There are also several experimental treatment protocols that involve exvivo activation and expansion of antigen specific T cells and adoptivetransfer of these cells into recipients in order to antigen-specific Tcells against tumor (Greenberg, R. et al., Science, 285:546-551 (1999)).These methods may also be used to activate T cell responses toinfectious agents such as CMV. Ex vivo activation in the presence ofmacrocyclic peptides may be expected to increase the frequency andactivity of the adoptively transferred T cells.

Infectious Diseases

Other methods of the disclosure are used to treat patients that havebeen exposed to particular toxins or pathogens. Accordingly, anotheraspect of the disclosure provides a method of treating an infectiousdisease in a subject comprising administering to the subject amacrocyclic peptide of the present disclosure such that the subject istreated for the infectious disease.

Similar to its application to tumors as discussed above, PD-L1 blockadecan be used alone, or as an adjuvant, in combination with vaccines, tostimulate the immune response to pathogens, toxins, and self-antigens.Examples of pathogens for which this therapeutic approach may beparticularly useful, include pathogens for which there is currently noeffective vaccine, or pathogens for which conventional vaccines are lessthan completely effective. These include, but are not limited to HIV,Hepatitis (A, B, and C), Influenza, Herpes, Giardia, Malaria (Butler, N.S. et al., Nature Immunology 13, 188-195 (2012); Hafalla, J. C. R., etal. PLOS Pathogens; Feb. 2, 2012)), Leishmania, Staphylococcus aureus,Pseudomonas Aeruginosa. PD-L1 blockade is particularly useful againstestablished infections by agents such as HIV that present alteredantigens over the course of the infections. These novel epitopes arerecognized as foreign at the time of anti-human PD-L1 administration,thus provoking a strong T cell response that is not dampened by negativesignals through PD-L1.

Some examples of pathogenic viruses causing infections treatable bymethods of the disclosure include HIV, hepatitis (A, B, or C), herpesvirus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus),adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus,coxsackie virus, cornovirus, respiratory syncytial virus, mumps virus,rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus,HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus,rabies virus, JC virus and arboviral encephalitis virus.

Some examples of pathogenic bacteria causing infections treatable bymethods of the disclosure include chlamydia, rickettsial bacteria,mycobacteria, staphylococci, streptococci, pneumonococci, meningococciand conococci, klebsiella, proteus, serratia, pseudomonas, legionella,diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax,plague, leptospirosis, and Lyme disease bacteria.

Some examples of pathogenic fungi causing infections treatable bymethods of the disclosure include Candida (albicans, krusei, glabrata,tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus,niger, etc.), Genus Mucorales(mucor, absidia, rhizophus), Sporothrixschenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis,Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites causing infections treatable bymethods of the disclosure include Entamoeba histolytica, Balantidiumcoli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia,Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesiamicroti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,Toxoplasma gondi, and Nippostrongylus brasiliensis.

In all of the above methods, PD-L1 blockade can be combined with otherforms of immunotherapy such as cytokine treatment (e.g., interferons,agents targeting VEGF activity or VEGF-receptors, GM-CSF, G-CSF, IL-2),or bispecific antibody therapy, which provides for enhanced presentationof tumor antigens (see, e.g., Holliger, Proc. Natl. Acad. Sci. USA,90:6444-6448 (1993); Poljak, Structure, 2:1121-1123 (1994)).

Autoimmune Reactions

The macrocyclic peptides may provoke and amplify autoimmune responses.Indeed, induction of anti-tumor responses using tumor cell and peptidevaccines reveals that many anti-tumor responses involve anti-selfreactivities (depigmentation observed in anti-CTLA-4+GM-CSF-modified B16 melanoma in van Elsas et al. supra; depigmentation in Trp-2vaccinated mice (Overwijk, W. et al., Proc. Natl. Acad. Sci. USA,96:2982-2987 (1999)); autoimmune prostatitis evoked by TRAMP tumor cellvaccines (Hurwitz, A., supra (2000)), melanoma peptide antigenvaccination and vitiligo observed in human clinical trials (Rosenberg,S. A. et al., J. Immunother. Emphasis Tumor Immunol., 19(1):81-84(1996)).

Therefore, it is possible to consider using anti-PD-L1 blockade inconjunction with various self proteins in order to devise vaccinationprotocols to efficiently generate immune responses against these selfproteins for disease treatment. For example, Alzheimer's diseaseinvolves inappropriate accumulation of A.beta. peptide in amyloiddeposits in the brain; antibody responses against amyloid are able toclear these amyloid deposits (Schenk et al., Nature, 400:173-177(1999)).

Other self proteins may also be used as targets such as IgE for thetreatment of allergy and asthma, and TNF.alpha for rheumatoid arthritis.Finally, antibody responses to various hormones may be induced by theuse of the macrocycles disclosed herein. Neutralizing antibody responsesto reproductive hormones may be used for contraception. Neutralizingantibody response to hormones and other soluble factors that arerequired for the growth of particular tumors may also be considered aspossible vaccination targets.

Analogous methods as described above for the use of anti-PD-L1macrocycles can be used for induction of therapeutic autoimmuneresponses to treat patients having an inappropriate accumulation ofother self-antigens, such as amyloid deposits, including A.beta. inAlzheimer's disease, cytokines such as TNF.alpha., and IgE.

Vaccines

The macrocyclic peptides may be used to stimulate antigen-specificimmune responses by coadministration of an anti-PD-1 macrocycle with anantigen of interest (e.g., a vaccine). Accordingly, in another aspectthe disclosure provides a method of enhancing an immune response to anantigen in a subject, comprising administering to the subject: (i) theantigen; and (ii) an anti-PD-1 macrocycle such that an immune responseto the antigen in the subject is enhanced. The antigen can be, forexample, a tumor antigen, a viral antigen, a bacterial antigen or anantigen from a pathogen. Non-limiting examples of such antigens includethose discussed in the sections above, such as the tumor antigens (ortumor vaccines) discussed above, or antigens from the viruses, bacteriaor other pathogens described above.

Suitable routes of administering the compositions (e.g., macrocyclicpeptides, multispecific and bispecific molecules and immunoconjugates)of the disclosure in vivo and in vitro are well known in the art and canbe selected by those of ordinary skill. For example, the compositionscan be administered by injection (e.g., intravenous or subcutaneous).Suitable dosages of the molecules used will depend on the age and weightof the subject and the concentration and/or formulation of thecomposition.

As previously described the macrocyclic peptides of the disclosure canbe co-administered with one or other more therapeutic agents, e.g., acytotoxic agent, a radiotoxic agent or an immunosuppressive agent. Thepeptide can be linked to the agent (as an immunocomplex) or can beadministered separate from the agent. In the latter case (separateadministration), the peptide can be administered before, after orconcurrently with the agent or can be co-administered with other knowntherapies, e.g., an anti-cancer therapy, e.g., radiation. Suchtherapeutic agents include, among others, anti-neoplastic agents such asdoxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine,chlorambucil, decarbazine and cyclophosphamide hydroxyurea which, bythemselves, are only effective at levels which are toxic or subtoxic toa patient. Cisplatin is intravenously administered as a 100 mg/dose onceevery four weeks and adriamycin is intravenously administered as a 60-75mg/ml dose once every 21 days. Co-administration of the macrocyclicpeptides of the present disclosure with chemotherapeutic agents providestwo anti-cancer agents which operate via different mechanisms whichyield a cytotoxic effect to human tumor cells. Such co-administrationcan solve problems due to development of resistance to drugs or a changein the antigenicity of the tumor cells which would render themunreactive with the peptides.

Also within the scope of the present disclosure are kits comprising thecompositions of the disclosure (e.g., macrocyclic peptides, bispecificor multispecific molecules, or immunoconjugates) and instructions foruse. The kit can further contain at least one additional reagent, or oneor more additional macrocyclic peptides of the disclosure (e.g., a humanantibody having a complementary activity which binds to an epitope inPD-L1 antigen distinct from the macrocycle). Kits typically include alabel indicating the intended use of the contents of the kit. The termlabel includes any writing, or recorded material supplied on or with thekit, or which otherwise accompanies the kit.

Combination Therapy

The combination of the macrocyclic peptides of the present disclosurewith another PD-L1 antagonist and/or other immunomodulator is useful forenhancement of an immune response against a hyperproliferative disease.For example, these molecules can be administered to cells in culture, invitro or ex vivo, or to human subjects, e.g., in vivo, to enhanceimmunity in a variety of situations. Accordingly, in one aspect, thedisclosure provides a method of modifying an immune response in asubject comprising administering to the subject a macrocyclic peptide ofthe disclosure such that the immune response in the subject is modified.Preferably, the response is enhanced, stimulated or up-regulated. Inanother embodiment, the instant disclosure provides a method of alteringadverse events associated with treatment of a hyperproliferative diseasewith an immunostimulatory therapeutic agent, comprising administering amacrocyclic peptide of the present disclosure and a subtherapeutic doseof another immunomodulator to a subject.

Blockade of PD-L1 by macrocyclic peptides can enhance the immuneresponse to cancerous cells in the patient. Cancers whose growth may beinhibited using the macrocyclic peptides of the instant disclosureinclude cancers typically responsive to immunotherapy. Representativeexamples of cancers for treatment with the combination therapy of theinstant disclosure include melanoma (e.g., metastatic malignantmelanoma), renal cancer, prostate cancer, breast cancer, colon cancerand lung cancer. Examples of other cancers that may be treated using themethods of the instant disclosure include bone cancer, pancreaticcancer, skin cancer, cancer of the head or neck, cutaneous orintraocular malignant melanoma, uterine cancer, ovarian cancer, rectalcancer, cancer of the anal region, stomach cancer, testicular cancer,uterine cancer, carcinoma of the fallopian tubes, carcinoma of theendometrium, carcinoma of the cervix, carcinoma of the vagina, carcinomaof the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, chronic or acute leukemias including acutemyeloid leukemia, chronic myeloid leukemia, acute lymphoblasticleukemia, chronic lymphocytic leukemia, solid tumors of childhood,lymphocytic lymphoma, cancer of the bladder, cancer of the kidney orureter, carcinoma of the renal pelvis, neoplasm of the central nervoussystem (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axistumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally induced cancers including those induced by asbestos, andcombinations of said cancers. The present disclosure is also useful fortreatment of metastatic cancers.

In certain embodiments, the combination of therapeutic agents containingat least one macrocyclic peptide discussed herein may be administeredconcurrently as a single composition in a pharmaceutically acceptablecarrier, or concurrently as separate compositions wherein each agent canbe administered sequentially. For example, a second immunomodulator anda macrocyclic peptide of the present disclosure can be administeredsequentially, such as the second immunomodulator administered first andthe macrocyclic peptide second, or the macrocyclic peptide beingadministered first and the second immunomodulator second. Furthermore,if more than one dose of the combination therapy is administeredsequentially, the order of the sequential administration can be reversedor kept in the same order at each time point of administration,sequential administrations may be combined with concurrentadministrations, or any combination thereof. For example, the firstadministration of a second immunomodulator and the macrocyclic peptidemay be concurrent, the second administration may be sequential with thesecond immunomodulator first and the macrocyclic peptide second, and thethird administration may be sequential with the macrocyclic peptidefirst and second immunomodulator second, etc. Another representativedosing scheme may involve a first administration that is sequential withthe macrocyclic peptide first and the second immunomodulator second, andsubsequent administrations may be concurrent.

Optionally, the combination of the macrocyclic peptide and a secondimmunomodulator can be further combined with an immunogenic agent, suchas cancerous cells, purified tumor antigens (including recombinantproteins, peptides, and carbohydrate molecules), cells, and cellstransfected with genes encoding immune stimulating cytokines (He et al.,J. Immunol., 173:4919-4928 (2004)). Non-limiting examples of tumorvaccines that can be used include peptides of melanoma antigens, such aspeptides of gp100, MAGE antigens, Trp-2, MART1 and/or tyrosinase, ortumor cells transfected to express the cytokine GM-CSF (discussedfurther below).

A combined PD-L1 macrocyclic peptide and a second immunomodulator can befurther combined with a vaccination protocol. Many experimentalstrategies for vaccination against tumors have been devised (seeRosenberg, S., Development of Cancer Vaccines, ASCO Educational BookSpring: 60-62 (2000); Logothetis, C., ASCO Educational Book Spring:300-302 (2000); Khayat, D., ASCO Educational Book Spring: 414-428(2000); Foon, K., ASCO Educational Book Spring: 730-738 (2000); see alsoRestifo et al., Cancer Vaccines, Chapter 61, pp. 3023-3043 in DeVita etal., eds., Cancer: Principles and Practice of Oncology, Fifth Edition(1997)). In one of these strategies, a vaccine is prepared usingautologous or allogeneic tumor cells. These cellular vaccines have beenshown to be most effective when the tumor cells are transduced toexpress GM-CSF. GM-CSF has been shown to be a potent activator ofantigen presentation for tumor vaccination (Dranoff et al., Proc. Natl.Acad. Sci. USA, 90:3539-3543 (1993)).

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so called tumor specificantigens (Rosenberg, Immunity, 10:281-287 (1999)). In many cases, thesetumor specific antigens are differentiation antigens expressed in thetumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens, and Trp-2. More importantly,many of these antigens can be shown to be the targets of tumor specificT cells found in the host. In certain embodiments, a combined PD-L1macrocyclic peptide and a second immunomodulator may be used inconjunction with a collection of recombinant proteins and/or peptidesexpressed in a tumor in order to generate an immune response to theseproteins. These proteins are normally viewed by the immune system asself-antigens and are, therefore, tolerant to them. The tumor antigenmay also include the protein telomerase, which is required for thesynthesis of telomeres of chromosomes and which is expressed in morethan 85% of human cancers and in only a limited number of somatictissues (Kim et al., Science, 266:2011-2013 (1994)). (These somatictissues may be protected from immune attack by various means). Tumorantigen may also be “neo-antigens” expressed in cancer cells because ofsomatic mutations that alter protein sequence or create fusion proteinsbetween two unrelated sequences (i.e., bcr-abl in the Philadelphiachromosome), or idiotype from B cell tumors.

Other tumor vaccines may include the proteins from viruses implicated inhuman cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses(HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). Another form oftumor specific antigen which may be used in conjunction with PD-L1macrocyclic peptide blockade is purified heat shock proteins (HSP)isolated from the tumor tissue itself. These heat shock proteins containfragments of proteins from the tumor cells and these HSPs are highlyefficient at delivery to antigen presenting cells for eliciting tumorimmunity (Suot et al., Science, 269:1585-1588 (1995); Tamura et al.,Science, 278:117-120 (1997)).

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DC's can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle et al., Nat. Med., 4:328-332 (1998)). DCs may alsobe transduced by genetic means to express these tumor antigens as well.DCs have also been fused directly to tumor cells for the purposes ofimmunization (Kugler et al., Nat. Med., 6:332-336 (2000)). As a methodof vaccination, DC immunization may be effectively further combined witha combined anti-PD-L1 macrocyclic peptide and a second immunomodulatorto activate more potent anti-tumor responses.

A combined anti-PD-L1 macrocyclic peptide and additional immunomodulatormay also be further combined with standard cancer treatments. Forexample, a combination of a macrocyclic peptide and a secondimmunomodulator may be effectively combined with chemotherapeuticregimes. In these instances, as is observed with the combination of amacrocyclic peptide and a second immunomodulator, it may be possible toreduce the dose of other chemotherapeutic reagent administered with thecombination of the instant disclosure (Mokyr et al., Cancer Res.,58:5301-5304 (1998)). An example of such a combination is a combinationof a macrocyclic peptide and a second immunomodulator further incombination with decarbazine for the treatment of melanoma. Anotherexample is a combination of a macrocyclic peptide and a secondimmunomodulatory agent further in combination with interleukin-2 (IL-2)for the treatment of melanoma. The scientific rationale behind thecombined use of PD-L1 macrocyclic peptide and another immunomodulatorwith chemotherapy is that cell death, which is a consequence of thecytotoxic action of most chemotherapeutic compounds, should result inincreased levels of tumor antigen in the antigen presentation pathway.Other combination therapies that may result in synergy with a combinedanti-PD-L1 macrocyclic peptide and additional immunomodulator throughcell death include radiation, surgery, or hormone deprivation. Each ofthese protocols creates a source of tumor antigen in the host.Angiogenesis inhibitors may also be combined with a combined PD-L1 andsecond immunomodulator. Inhibition of angiogenesis leads to tumor celldeath, which may also be a source of tumor antigen to be fed into hostantigen presentation pathways.

A combination of PD-L1 and another immunomodulator can also be used incombination with bispecific macrocyclic peptides that target Fc.alpha.or Fc.gamma. receptor-expressing effector cells to tumor cells (see,e.g., U.S. Pat. Nos. 5,922,845 and 5,837,243). Bispecific macrocyclicpeptides can be used to target two separate antigens. For exampleanti-Fc receptor/anti tumor antigen (e.g., Her-2/neu) bispecificmacrocyclic peptides have been used to target macrophages to sites oftumor. This targeting may more effectively activate tumor specificresponses. The T cell arm of these responses would be augmented by theuse of a combined PD-L1 and a second immunomodulator. Alternatively,antigen may be delivered directly to DCs by the use of bispecificmacrocyclic peptides which bind to tumor antigen and a dendritic cellspecific cell surface marker.

In another example, a combination of a macrocyclic peptide and a secondimmunomodulator can be used in conjunction with anti-neoplasticmacrocyclic agents, such as RITUXAN® (rituximab), HERCEPTIN®(trastuzumab), BEXXAR® (tositumomab), ZEVALIN® (ibritumomab), CAMPATH®(alemtuzumab), Lymphocide (eprtuzumab), AVASTIN® (bevacizumab), andTARCEVA® (erlotinib), and the like. By way of example and not wishing tobe bound by theory, treatment with an anti-cancer antibody or ananti-cancer antibody conjugated to a toxin can lead to cancer cell death(e.g., tumor cells) which would potentiate an immune response mediatedby the second immunomodulator target or PD-L1. In an exemplaryembodiment, a treatment of a hyperproliferative disease (e.g., a cancertumor) may include an anti-cancer antibody in combination with amacrocyclic peptide and a second immunomodulator concurrently orsequentially or any combination thereof, which may potentiate ananti-tumor immune responses by the host.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation ofproteins, which are expressed by the tumors and which areimmunosuppressive. These include, among others, TGF-.beta. (Kehrl, J. etal., J. Exp. Med., 163:1037-1050 (1986)), IL-10 (Howard, M. et al.,Immunology Today, 13:198-200 (1992)), and Fas ligand (Hahne, M. et al.,Science, 274:1363-1365 (1996)). In another example, antibodies to eachof these entities may be further combined with a macrocyclic peptide andanother immunomodulator to counteract the effects of immunosuppressiveagents and favor anti-tumor immune responses by the host.

Other agents that may be used to activate host immune responsiveness canbe further used in combination with a macrocyclic peptide of the presentdisclosure. These include molecules on the surface of dendritic cellsthat activate DC function and antigen presentation. Anti-CD40macrocyclic peptides are able to substitute effectively for T cellhelper activity (Ridge, J. et al., Nature, 393:474-478 (1998)) and canbe used in conjunction with the macrocyclic peptides of the presentdisclosure, either alone or in combination with an anti-CTLA-4combination (Ito, N. et al., Immunobiology, 201(5):527-540 (2000)).Activating macrocyclic peptides to T cell costimulatory molecules, suchas OX-40 (Weinberg, A. et al., Immunol., 164:2160-2169 (2000)), 4-1BB(Melero, I. et al., Nat. Med., 3:682-685 (1997), and ICOS (Hutloff, A.et al., Nature, 397:262-266 (1999)) may also provide for increasedlevels of T cell activation.

Bone marrow transplantation is currently being used to treat a varietyof tumors of hematopoietic origin. While graft versus host disease is aconsequence of this treatment, therapeutic benefit may be obtained fromgraft vs. tumor responses. A macrocyclic peptide of the presentdisclosure, either alone or in combination with another immunomodulator,can be used to increase the effectiveness of the donor engrafted tumorspecific T cells.

There are also several experimental treatment protocols that involve exvivo activation and expansion of antigen specific T cells and adoptivetransfer of these cells into recipients in order to antigen-specific Tcells against tumor (Greenberg, R. et al., Science, 285:546-551 (1999)).These methods may also be used to activate T cell responses toinfectious agents such as CMV. Ex vivo activation in the presence amacrocyclic peptide of the present disclosure, either alone or incombination with another innumomodulator, may be expected to increasethe frequency and activity of the adoptively transferred T cells.

In certain embodiments, the present disclosure provides a method foraltering an adverse event associated with treatment of ahyperproliferative disease with an immunostimulatory agent, comprisingadministering a macrocyclic peptide of the present disclosure incombination with a subtherapeutic dose of another immunomodulator to asubject. For example, the methods of the present disclosure provide fora method of reducing the incidence of immunostimulatory therapeuticantibody-induced colitis or diarrhea by administering a non-absorbablesteroid to the patient. Because any patient who will receive animmunostimulatory therapeutic antibody is at risk for developing colitisor diarrhea induced by such treatment, this entire patient population issuitable for therapy according to the methods of the present disclosure.Although steroids have been administered to treat inflammatory boweldisease (IBD) and prevent exacerbations of IBD, they have not been usedto prevent (decrease the incidence of) IBD in patients who have not beendiagnosed with IBD. The significant side effects associated withsteroids, even non-absorbable steroids, have discouraged prophylacticuse.

In further embodiments, a macrocyclic peptide of the present disclosure,either alone or in combination with another immunomodulator, can befurther combined with the use of any non-absorbable steroid. As usedherein, a “non-absorbable steroid” is a glucocorticoid that exhibitsextensive first pass metabolism such that, following metabolism in theliver, the bioavailability of the steroid is low, i.e., less than about20%. In one embodiment of the disclosure, the non-absorbable steroid isbudesonide. Budesonide is a locally-acting glucocorticosteroid, which isextensively metabolized, primarily by the liver, following oraladministration. ENTOCORT® EC (Astra-Zeneca) is a pH- and time-dependentoral formulation of budesonide developed to optimize drug delivery tothe ileum and throughout the colon. ENTOCORT® EC is approved in the U.S.for the treatment of mild to moderate Crohn's disease involving theileum and/or ascending colon. The usual oral dosage of ENTOCORT® EC forthe treatment of Crohn's disease is 6 to 9 mg/day. ENTOCORT® EC isreleased in the intestines before being absorbed and retained in the gutmucosa. Once it passes through the gut mucosa target tissue, ENTOCORT®EC is extensively metabolized by the cytochrome P450 system in the liverto metabolites with negligible glucocorticoid activity. Therefore, thebioavailability is low (about 10%). The low bioavailability ofbudesonide results in an improved therapeutic ratio compared to otherglucocorticoids with less extensive first-pass metabolism. Budesonideresults in fewer adverse effects, including less hypothalamic-pituitarysuppression, than systemically-acting corticosteroids. However, chronicadministration of ENTOCORT® EC can result in systemic glucocorticoideffects such as hypercorticism and adrenal suppression. See Physicians'Desk Reference Supplement, 58th Edition, 608-610 (2004).

In still further embodiments, a combination PD-L1 and anotherimmunomodulator in conjunction with a non-absorbable steroid can befurther combined with a salicylate. Salicylates include 5-ASA agentssuch as, for example: sulfasalazine (AZULFIDINE®, Pharmacia & Upjohn);olsalazine (DIPENTUM®, Pharmacia & UpJohn); balsalazide (COLAZAL®, SalixPharmaceuticals, Inc.); and mesalamine (ASACOL®, Procter & GamblePharmaceuticals; PENTASA®, Shire US; CANASA®, Axcan Scandipharm, Inc.;ROWASA®, Solvay).

Dosage and Formulation

A suitable peptide of Formula I, or more specifically a macrocyclicpeptide described herein, can be administered to patients to treatdiabetes and other related diseases as the compound alone and or mixedwith an acceptable carrier in the form of pharmaceutical formulations.Those skilled in the art of treating diabetes can easily determine thedosage and route of administration of the compound to mammals, includinghumans, in need of such treatment. The route of administration mayinclude but is not limited to oral, intraoral, rectal, transdermal,buccal, intranasal, pulmonary, subcutaneous, intramuscular, intradermal,sublingual, intracolonic, intraoccular, intravenous, or intestinaladministration. The compound is formulated according to the route ofadministration based on acceptable pharmacy practice (Fingl et al., inThe Pharmacological Basis of Therapeutics, Chapter 1, p. 1 (1975);Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co.,Easton, Pa. (1990)).

The pharmaceutically acceptable peptide compositions described hereincan be administered in multiple dosage forms such as tablets, capsules(each of which includes sustained release or timed releaseformulations), pills, powders, granules, elixirs, in situ gels,microspheres, crystalline complexes, liposomes, micro-emulsions,tinctures, suspensions, syrups, aerosol sprays and emulsions. Thecompositions described herein can also be administered in oral,intravenous (bolus or infusion), intraperitoneal, subcutaneous,transdermally or intramuscular form, all using dosage forms well knownto those of ordinary skill in the pharmaceutical arts. The compositionsmay be administered alone, but generally will be administered with apharmaceutical carrier selected on the basis of the chosen route ofadministration and standard pharmaceutical practice.

The dosage regimen for the compositions described herein will, ofcourse, vary depending upon known factors, such as the pharmacodynamiccharacteristics of the particular agent and its mode and route ofadministration; the species, age, sex, health, medical condition, andweight of the recipient; the nature and extent of the symptoms; the kindof concurrent treatment; the frequency of treatment; the route ofadministration, the renal and hepatic function of the patient, and theeffect desired. A physician or veterinarian can determine and prescribethe effective amount of the drug required to prevent, counter, or arrestthe progress of the disease state.

By way of general guidance, the daily oral dosage of the activeingredient, when used for the indicated effects, will range betweenabout 0.001 to 1000 mg/kg of body weight, preferably between about 0.01to 100 mg/kg of body weight per day, and most preferably between about0.6 to 20 mg/kg/day. Intravenously, the daily dosage of the activeingredient when used for the indicated effects will range between 0.001ng to 100.0 ng per min/per Kg of body weight during a constant rateinfusion. Such constant intravenous infusion can be preferablyadministered at a rate of 0.01 ng to 50 ng per min per Kg body weightand most preferably at 0.01 ng to 10.0 mg per min per Kg body weight.The compositions described herein may be administered in a single dailydose, or the total daily dosage may be administered in divided doses oftwo, three, or four times daily. The compositions described herein mayalso be administered by a depot formulation that will allow sustainedrelease of the drug over a period of days/weeks/months as desired.

The compositions described herein can be administered in intranasal formvia topical use of suitable intranasal vehicles, or via transdermalroutes, using transdermal skin patches. When administered in the form ofa transdermal delivery system, the dosage administration will, ofcourse, be continuous rather than intermittent throughout the dosageregimen.

The compositions are typically administered in a mixture with suitablepharmaceutical diluents, excipients, or carriers (collectively referredto herein as pharmaceutical carriers) suitably selected with respect tothe intended form of administration, that is, oral tablets, capsules,elixirs, aerosol sprays generated with or without propellant and syrups,and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic, pharmaceutically acceptable, inert carrier such as but notlimited to, lactose, starch, sucrose, glucose, methyl cellulose,magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, andsorbitol; for oral administration in liquid form, the oral drugcomponents can be combined with any oral, non-toxic, pharmaceuticallyacceptable inert carrier such as, but not limited to, ethanol, glycerol,and water. Moreover, when desired or necessary, suitable binders,lubricants, disintegrating agents, and coloring agents can also beincorporated into the mixture. Suitable binders include, but not limitedto, starch, gelatin, natural sugars such as, but not limited to, glucoseor beta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth, or sodium alginate, carboxymethylcellulose,polyethylene glycol, and waxes. Lubricants used in these dosage formsinclude sodium oleate, sodium stearate, magnesium stearate, sodiumbenzoate, sodium acetate, and sodium chloride. Disintegrants include,but are not limited to, starch, methyl cellulose, agar, bentonite, andxanthan gum.

The compositions described herein may also be administered in the formof mixed micellar or liposome delivery systems, such as smallunilamellar vesicles, large unilamellar vesicles, and multilamellarvesicles. Liposomes can be formed from a variety of phospholipids, suchas cholesterol, stearylamine, or phosphatidylcholines. Permeationenhancers may be added to enhance drug absorption.

Since prodrugs are known to enhance numerous desirable qualities ofpharmaceuticals (i.e., solubility, bioavailability, manufacturing, etc.)the compounds described herein may be delivered in prodrug form. Thus,the subject matter described herein is intended to cover prodrugs of thepresently claimed compounds, methods of delivering the same, andcompositions containing the same.

The compositions described herein may also be coupled with solublepolymers as targetable drug carriers. Such polymers can includepolyvinyl-pyrrolidone, pyran copolymer,polyhydroxypropyl-methacrylamide-phenol,polyhydroxyethylaspartamidephenol, or polyethyleneoxide-polyly sinesubstituted with palmitoyl residues. Furthermore, the compositionsdescribed herein may be combined with a class of biodegradable polymersuseful in achieving controlled release of a drug, for example,polylactic acid, polyglycolic acid, copolymers of polylactic andpolyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, andcrosslinked or amphipathic block copolymers of hydrogels.

Dosage forms (pharmaceutical compositions) suitable for administrationmay contain from about 0.01 milligram to about 500 milligrams of activeingredient per dosage unit. In these pharmaceutical compositions theactive ingredient will ordinarily be present in an amount of about0.5-95% by weight based on the total weight of the composition.

Gelatin capsules may contain the active ingredient and powderedcarriers, such as lactose, starch, cellulose derivative, magnesiumstearate, and stearic acid. Similar diluents can be used to makecompressed tablets. Both tablets and capsules can be manufactured assustained release products to provide for continuous release ofmedication over a period of hours. Compressed tablets can be sugarcoated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric coated for selectivedisintegration in the gastrointestinal tract.

Liquid dosage forms for oral administration can contain coloring andflavoring to increase patient acceptance.

In general, water, a suitable oil, saline, aqueous dextrose (glucose),and related sugar solutions and glycols such as propylene glycol orpolyethylene glycols are suitable carriers for parenteral solutions.Solution for parenteral administration preferably contains awater-soluble salt of the active ingredient, suitable stabilizingagents, and if necessary, buffer substances. Antioxidizing agents suchas sodium bisulfite, sodium sulfite, or ascorbic acid, either alone orcombined, are suitable stabilizing agents. Also used are citric acid andits salts and sodium EDTA. In addition, parenteral solutions can containpreservatives, such as benzalkonium chloride, methyl- or propyl-paraben,and chlorobutanol.

Suitable pharmaceutical carriers are described in Remington: The Scienceand Practice of Pharmacy, Nineteenth Edition, Mack Publishing Company(1995), a standard reference text in this field.

Representative useful pharmaceutical dosage forms for administration ofthe compounds described herein can be illustrated as follows:

Capsules

A large number of unit capsules can be prepared by filling standardtwo-piece hard gelatin capsules with 100 milligrams of powdered activeingredient, 150 milligrams of lactose, 50 milligrams of cellulose, and 6milligrams magnesium stearate.

Soft Gelatin Capsules

A mixture of active ingredient in a digestible oil such as soybean oil,cottonseed oil or olive oil may be prepared and injected by means of apositive displacement pump into gelatin to form soft gelatin capsulescontaining 100 milligrams of the active ingredient. The capsules shouldbe washed and dried.

Tablets

Tablets may be prepared by conventional procedures so that the dosageunit, for example is 100 milligrams of active ingredient, 0.2 milligramsof colloidal silicon dioxide, 5 milligrams of magnesium stearate, 275milligrams of microcrystalline cellulose, 11 milligrams of starch and98.8 milligrams of lactose. Appropriate coatings may be applied toincrease palatability or delay absorption.

Injectable

An injectable formulation of a peptide composition described herein mayor may not require the use of excipients such as those that have beenapproved by regulatory bodies. These excipients include, but are notlimited to, solvents and co-solvents, solubilizing, emulsifying orthickening agents, chelating agents, antioxidants and reducing agents,antimicrobial preservatives, buffers and pH adjusting agents, bulkingagents, protectants and tonicity adjustors and special additives. Aninjectable formulation has to be sterile, pyrogen free and, in the caseof solutions, free of particulate matter.

A parenteral composition suitable for administration by injection may beprepared by stirring for example, 1.5% by weight of active ingredient ina pharmaceutically acceptable buffer that may or may not contain aco-solvent or other excipient. The solution should be made isotonic withsodium chloride and sterilized.

Suspension

An aqueous suspension can be prepared for oral and/or parenteraladministration so that, for example, each 5 mL contains 100 mg of finelydivided active ingredient, 20 mg of sodium carboxymethyl cellulose, 5 mgof sodium benzoate, 1.0 g of sorbitol solution, U.S.P., and 0.025 mL ofvanillin or other palatable flavoring.

Biodegradable Microparticles

A sustained-release parenteral composition suitable for administrationby injection may be prepared, for example, by dissolving a suitablebiodegradable polymer in a solvent, adding to the polymer solution theactive agent to be incorporated, and removing the solvent from thematrix thereby forming the matrix of the polymer with the active agentdistributed throughout the matrix.

Peptide Synthesis

The description of the present disclosure herein should be construed incongruity with the laws and principals of chemical bonding. It should beunderstood that the compounds encompassed by the present disclosure arethose that are suitably stable for use as pharmaceutical agent. One ofskill in the art will know what compounds would and would not be stablebased on the general principles of chemical bonding and stability.

Chemical synthesis of a macrocyclic peptide of the present disclosurecan be carried out using a variety of art recognized methods, includingstepwise solid phase synthesis, semi-synthesis through theconformationally-assisted re-ligation of peptide fragments, enzymaticligation of cloned or synthetic peptide segments, and chemical ligation.A preferred method to synthesize the macrocyclic peptides and analogsthereof described herein is chemical synthesis using various solid-phasetechniques such as those described in Chan, W. C. et al., eds., FmocSolid Phase Synthesis, Oxford University Press, Oxford (2000); Barany,G. et al., The Peptides: Analysis, Synthesis, Biology, Vol. 2: “SpecialMethods in Peptide Synthesis, Part A”, pp. 3-284, Gross, E. et al.,eds., Academic Press, New York (1980); and in Stewart, J. M. et al.,Solid-Phase Peptide Synthesis, 2nd Edition, Pierce Chemical Co.,Rockford, Ill. (1984). The preferred strategy is based on the Fmoc(9-Fluorenylmethyl methyl-oxycarbonyl) group for temporary protection ofthe α-amino group, in combination with the tert-butyl group fortemporary protection of the amino acid side chains (see for exampleAtherton, E. et al., “The Fluorenylmethoxycarbonyl Amino ProtectingGroup”, in The Peptides: Analysis, Synthesis, Biology, Vol. 9: “SpecialMethods in Peptide Synthesis, Part C”, pp. 1-38, Undenfriend, S. et al.,eds., Academic Press, San Diego (1987).

The peptides can be synthesized in a stepwise manner on an insolublepolymer support (also referred to as “resin”) starting from theC-terminus of the peptide. A synthesis is begun by appending theC-terminal amino acid of the peptide to the resin through formation ofan amide or ester linkage. This allows the eventual release of theresulting peptide as a C-terminal amide or carboxylic acid,respectively.

The C-terminal amino acid and all other amino acids used in thesynthesis are required to have their α-amino groups and side chainfunctionalities (if present) differentially protected such that theα-amino protecting group may be selectively removed during thesynthesis. The coupling of an amino acid is performed by activation ofits carboxyl group as an active ester and reaction thereof with theunblocked α-amino group of the N-terminal amino acid appended to theresin. The sequence of α-amino group deprotection and coupling isrepeated until the entire peptide sequence is assembled. The peptide isthen released from the resin with concomitant deprotection of the sidechain functionalities, usually in the presence of appropriate scavengersto limit side reactions. The resulting peptide is finally purified byreverse phase HPLC.

The synthesis of the peptidyl-resins required as precursors to the finalpeptides utilizes commercially available cross-linked polystyrenepolymer resins (Novabiochem, San Diego, Calif.; Applied Biosystems,Foster City, Calif.). Preferred solid supports are:4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetyl-p-methylbenzhydrylamine resin (Rink amide MBHA resin);9-Fmoc-amino-xanthen-3-yloxy-Merrifield resin (Sieber amide resin);4-(9-Fmoc)aminomethyl-3,5-dimethoxyphenoxy)valeryl-aminomethyl-Merrifieldresin (PAL resin), for C-terminal carboxamides. Coupling of first andsubsequent amino acids can be accomplished using HOBt, 6-Cl-HOBt or HOAtactive esters produced from DIC/HOBt, HBTU/HOBt, BOP, PyBOP, or fromDIC/6-Cl-HOBt, HCTU, DIC/HOAt or HATU, respectively. Preferred solidsupports are: 2-Chlorotrityl chloride resin and9-Fmoc-amino-xanthen-3-yloxy-Merrifield resin (Sieber amide resin) forprotected peptide fragments. Loading of the first amino acid onto the2-chlorotrityl chloride resin is best achieved by reacting theFmoc-protected amino acid with the resin in dichloromethane and DIEA. Ifnecessary, a small amount of DMF may be added to facilitate dissolutionof the amino acid.

The syntheses of the peptide analogs described herein can be carried outby using a single or multi-channel peptide synthesizer, such as an CEMLiberty Microwave synthesizer, or a Protein Technologies, Inc. Prelude(6 channels) or Symphony (12 channels) synthesizer.

The peptidyl-resin precursors for their respective peptides may becleaved and deprotected using any standard procedure (see, for example,King, D. S. et al., Int. J. Peptide Protein Res., 36:255-266 (1990)). Adesired method is the use of TFA in the presence of water and TIS asscavengers. Typically, the peptidyl-resin is stirred in TFA/water/TIS(94:3:3, v:v:v; 1 mL/100 mg of peptidyl resin) for 2-6 hrs at roomtemperature. The spent resin is then filtered off and the TFA solutionis concentrated or dried under reduced pressure. The resulting crudepeptide is either precipitated and washed with Et₂O or is redissolveddirectly into DMSO or 50% aqueous acetic acid for purification bypreparative HPLC.

Peptides with the desired purity can be obtained by purification usingpreparative HPLC, for example, on a Waters Model 4000 or a ShimadzuModel LC-8A liquid chromatograph. The solution of crude peptide isinjected into a YMC S5 ODS (20×100 mm) column and eluted with a lineargradient of MeCN in water, both buffered with 0.1% TFA, using a flowrate of 14-20 mL/min with effluent monitoring by UV absorbance at 220nm. The structures of the purified peptides can be confirmed byelectro-spray MS analysis.

EXPERIMENTAL PROCEDURES

The abbreviations used in the present application, includingparticularly in the illustrative schemes and examples which follow, arewell-known to those skilled in the art. Some of the abbreviations usedare as follows: DMF for N,N-dimethylformamide; HATU forO-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate; HCTU forO-(6-C₁-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate;TFA for trifluoroacetic acid; DBU for1,8-diazobicyclo[5.4.0]undec-7-ene; DIAD for diisopropylazodicarboxylate; TIS for triisopropylsilane; DMSO fordimethylsulfoxide; MeCN or ACN for acetonitrile; DCM for1,1-dichloromethane; DIEA or DIPEA for diisopropylethylamine; Fmoc for9-fluorenylmethyloxycarbonyl; NMM for N-methylmorpholine; DMAP for4-N,N-dimethylaminopyridine; NMP for N-methylpyrrolidone; Ac for acetyl;and Et for ethyl.

Analytical Data:

Mass Spectrometry: “ESI-MS(+)” signifies electrospray ionization massspectrometry performed in positive ion mode; “ESI-MS(−)” signifieselectrospray ionization mass spectrometry performed in negative ionmode; “ESI-HRMS(+)” signifies high-resolution electrospray ionizationmass spectrometry performed in positive ion mode; “ESI-HRMS(−)”signifies high-resolution electrospray ionization mass spectrometryperformed in negative ion mode. The detected masses are reportedfollowing the “m/z” unit designation. Compounds with exact massesgreater than 1000 were often detected as double-charged ortriple-charged ions.

Analysis LCMS Condition A:

Column: Waters BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A:water with 0.05% TFA; Mobile Phase B: Acetonitrile with 0.05% TFA;Temperature: 50° C.; Gradient: 2% B to 98% B over 2 min., then a 0.5min. hold at 98% B; Flow: 0.8 mL/min; Detection: UV at 220 nm.

Analysis LCMS Condition C:

Column: Waters BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A:water with 0.2% Formic Acid and 0.01% TFA; Mobile Phase B: Acetonitrilewith 0.2% Formic acid an 0.01% TFA; Temperature: 50° C.; Gradient: 2% Bto 80% B over 2 min., 80% B to 98% B over 0.1 minute then a 0.5 min.hold at 98% B; Flow: 0.8 mL/min; Detection: UV at 220 nm.

Analysis LCMS Condition D:

Column: Waters BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A:5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 70°C.; Gradient: 0-100% B over 3 min., then a 2.0-minute hold at 100% B;Flow: 0.75 mL/min; Detection: UV at 220 nm.

Analysis LCMS Condition E:

Column: Waters BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A:5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B:95:5 acetonitrile:water with 0.1% trifluoroacetic acid; temperature: 50°C. or 70° C.; Gradient: 0-100% B over 3 min., then a 0.75 or 2.0-minutehold at 100% B; Flow: 0.75 or 1.11 mL/min; Detection: UV at 220 nm.

Analysis LCMS Condition F:

Column: Waters XBridge C18, 2.1×50 mm; Mobile Phase A: 5:95acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10 mM ammonium acetate; Temperature: 35° C.;Gradient: 0-100% B over 4 min., then a 1-minute hold at 100% B; Flow: 4mL/min; Detection: UV at 220 nm.

Analysis LCMS Condition G:

Column: Waters BEH C18, 2.0×50 mm, 1.7-μm particles; Mobile Phase A:5:95 methanol:water with 10 mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10 mM ammonium acetate; Temperature: 50° C.;Gradient: 0-100% B over 3 min., then a 0.5-minute hold at 100% B; Flow:0.5 mL/min; Detection: UV at 220 nm.

Analysis LCMS Condition H:

Column: Waters BEH C18, 2.0×50 mm, 1.7-μm particles; Mobile Phase A:5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 50°C.; Gradient: 0-100% B over 3 minutes, then a 0.5-minute hold at 100% B;Flow: 1.0 mL/min; Detection: UV at 220 nm.

Analysis LCMS Condition I:

Column: Waters BEH C18, 2.0×50 mm, 1.7-μm particles; Mobile Phase A:5:95 methanol:water with 10 mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10 mM ammonium acetate; Temperature: 50° C.;Gradient: 0-100% B over 3 minutes, then a 0.5-minute hold at 100% B;Flow: 0.5 mL/min; Detection: UV at 220 nm.

Analysis LCMS Condition J:

Waters CSH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: 5:95acetonitrile:water with trifluoroacetic acid; Mobile Phase B: 95:5acetonitrile:water with trifluoroacetic acid; Temperature: 70° C.;Gradient: 0% B, 0-100% B over 3 minutes, then a 2.0-minute hold at 100%B; Flow: 0.75 mL/min; Detection: UV at 220 nm.

General Procedures: Symphony Method A:

All manipulations were performed under automation on a Symphony peptidesynthesizer (Protein Technologies). All procedures unless noted wereperformed in a Symphony polypropylene vessel fitted with a bottom frit.The vessel connects to the Symphony peptide synthesizer through both thebottom and the top of the tube. All solvents, DMF, DCM, amino acids andreagents are added through the bottom of the vessel and pass up throughthe frit to contact the resin. All solutions are removed through thebottom of the vessel. “Periodic agitation” describes a brief pulse of N₂gas through the bottom frit; the pulse lasts approximately 5 seconds andoccurs every 15 seconds. Amino acid solutions were generally not usedbeyond three weeks from preparation. HATU solution was used within 5days of preparation. DMF=dimethylformamide; DCM=dichloromethane;THF=tetrahydrofuran;HCTU=2-(6-Chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium;HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate; NMIVI=n-Methyl morpholine;DIPEA=diisopropylethylamine; DMAP=N,N-dimethylpyridin-4-amine; Rinkresin=4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-aminomethylresin; Sieber resin=Fmoc-amino-xanthen-3-yloxy, where “3-yloxy”describes the position and type of connectivity to the polystyreneresin. The resins used are Merrifield polymer (polystyrene) with eithera Rink or a Sieber linker (Fmoc-protected at nitrogen); 100-200 mesh, 1%DVB, 0.35 mmol/g or 0.71 mmol/g loading, respectively. Other common acidsensitive resins can also be used in the synthesis such asfunctionalized Chlorotrityl Resin. Common amino acids used are listedbelow with side-chain protecting groups indicated inside parenthesis.Fmoc-Ala-OH; Fmoc-Arg(Pbf)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Asp(OtBu)-OH;Fmoc-Cys(Trt)-OH; Fmoc-Dab(Boc)-OH (Dab=2,4-diaminobutyric acid);Fmoc-Dap(Boc)-OH (Dap=2,3-diaminopropionic acid); Fmoc-Gln(Trt)-OH;Fmoc-Gly-OH; Fmoc-His(Trt)-OH; Fmoc-t-Hyp(tBu)-OH(t-Hyp=trans-4-hydroxyproline); Fmoc-Ile-OH; Fmoc-Leu-OH;Fmoc-Lys(Boc)-OH; Fmoc-Nle-OH; Fmoc-Met-OH; Fmoc-[N-Me]Ala-OH;Fmoc[N-Me]Nle-OH; Fmoc-Phe-OH; Fmoc-Pro-OH; Fmoc-Sar-OH (Sar=Sarcosineor [N-Me]Gly); Fmoc-Ser(tBu)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Trp(Boc)-OH;Fmoc-Tyr(tBu)-OH; Fmoc-Val-OH.

The procedures of “Symphony Method A” describe an experiment performedon a 0.050-0.100 mmol scale, where the scale is determined by the resinsubstitution. This scale corresponds to approximately 143-286 mg or70-140 mg of the Rink or Sieber-Merrifield resins, respectively,described above. All procedures can be scaled beyond the 0.050-0.100mmol scale by adjusting the described volumes by the multiple of thescale. Prior to amino acid coupling, all peptide synthesis sequencesbegan with a resin-swelling procedure, described below as “Swellingprocedure”. Coupling of amino acids to a primary amine N-terminus usedthe “Standard-coupling procedure” described below. Coupling of aminoacids to a secondary amine N-terminus used the “Secondary-aminecoupling” procedure described below.

Swelling Procedure:

To a Symphony polypropylene reaction vessel was added Merrifield Rink orSieber resin (70 mg, 0.050 mmol or 140 mg, 0.100 mmol). The resin waswashed (swelled) three times as follows: to the reaction vessel wasadded DMF (2.5-5.0 mL) upon which the mixture was periodically agitatedwith N₂ bubbling from the bottom of the reaction vessel for 10 minutesbefore the solvent was drained through the frit.

Standard-Coupling Procedure:

The resin was washed three times as follows: to the reaction vessel wasadded DMF (2.5-5.0 mL) upon which the mixture was periodically agitatedwith N₂ bubbling from the bottom of the reaction vessel for 30 secondsbefore the solvent was drained through the frit. To the reaction vesselwas added piperidine:DMF (20:80 v/v, 2.5-5.0 mL). The mixture wasperiodically agitated for 5 minutes and then the solution was drainedthrough the frit. The procedure was repeated one more time. The resinwas washed 6 times as follows: for each wash, DMF (2.5-5.0 mL) was addedthrough the bottom of the vessel and the resulting mixture wasperiodically agitated for 30 seconds before the solution was drainedthrough the frit. To the reaction vessel was added the amino acid (0.2Min DMF, 1.25-2.5 mL, 5 eq), then HATU (0.2M in DMF, 1.25-2.5 mL, 5 eq),and finally NMM (0.8M in DMF, 1.25-2.5 mL, 10 eq). The mixture wasperiodically agitated for one hour, then the reaction solution wasdrained through the frit. The resin was washed with DMF (2.5-5.0 mL)five times, stirring it for 30 seconds each time. To the reaction vesselwas added a solution of acetic anhydride:DIEA:DMF (10:5:85 v/v/v,2.5-5.0 mL). The mixture was periodically agitated for 20 min., then thesolution was drained through the frit. The resin was washed successivelyfive times as follows: for each wash, DMF (2.5-5.0 mL) was added throughthe top of the vessel and the resulting mixture was periodicallyagitated for 90 seconds before the solution was drained through thefrit. The resulting resin was used directly in the next step.

Secondary Amine-Coupling Procedure:

The resin was washed three times as follows: to the reaction vessel wasadded DMF (2.5-5.0 mL) upon which the mixture was periodically agitatedwith N₂ bubbling from the bottom of the reaction vessel for 30 secondsbefore the solvent was drained through the frit. To the reaction vesselwas added piperidine:DMF (20:80 v/v, 2.5-5.0 mL). The mixture wasperiodically agitated for 5 minutes and then the solution was drainedthrough the frit. The procedure was repeated one more time. The resinwas washed 6 times as follows: for each wash, DMF (2.5-5.0 mL) was addedthrough the bottom of the vessel and the resulting mixture wasperiodically agitated for 30 seconds before the solution was drainedthrough the frit. To the reaction vessel was added the amino acid (0.2Min DMF, 2.5-5.0 mL, 10 eq), then HATU (0.2M in DMF, 2.5-5.0 mL, 10 eq),and finally NMM (0.8M in DMF, 2.5-5.0 mL, 20 eq). The mixture wasperiodically agitated for two hours, then the reaction solution wasdrained through the frit. The resin was washed with DMF (2.5-5.0 mL)five times, stirring it for 30 seconds each time. To the reaction vesselwas added a solution of acetic anhydride:DIEA:DMF (10:5:85 v/v/v,2.5-5.0 mL). The mixture was periodically agitated for 20 min., then thesolution was drained through the frit. The resin was washed successivelyfive times as follows: for each wash, DMF (2.5-5.0 mL) was added throughthe top of the vessel and the resulting mixture was periodicallyagitated for 90 seconds before the solution was drained through thefrit. The resulting resin was used directly in the next step.

Secondary Amine-Coupling without Fmoc Deprotection Procedure:

The resin was washed three times as follows: to the reaction vessel wasadded DMF (2.5-5.0 mL) upon which the mixture was periodically agitatedwith N₂ bubbling from the bottom of the reaction vessel for 30 secondsbefore the solvent was drained through the frit. To the reaction vesselwas added the amino acid (0.2M in DMF, 2.5-5.0 mL, 10 eq), then HATU(0.2M in DMF, 2.5-5.0 mL, 10 eq), and finally NMM (0.8M in DMF, 2.5-5.0mL, 20 eq). The mixture was periodically agitated for six hours, thenthe reaction solution was drained through the frit. The resin was washedwith DMF (2.5-5.0 mL) five times, stirring it for 30 seconds each time.To the reaction vessel was added a solution of acetic anhydride:DIEA:DMF(10:5:85 v/v/v, 2.5-5.0 mL). The mixture was periodically agitated for20 min., then the solution was drained through the frit. The resin waswashed successively five times as follows: for each wash, DMF (2.5-5.0mL) was added through the top of the vessel and the resulting mixturewas periodically agitated for 90 seconds before the solution was drainedthrough the frit. The resulting resin was used directly in the nextstep.

Chloroacetic Anhydride Capping Procedure:

The resin was washed three times as follows: to the reaction vessel wasadded DMF (2.5-5.0 mL) upon which the mixture was periodically agitatedwith N₂ bubbling from the bottom of the reaction vessel for 30 secondsbefore the solvent was drained through the frit. To the reaction vesselwas added piperidine:DMF (20:80 v/v, 2.5-5.0 mL). The mixture wasperiodically agitated for 5 minutes and then the solution was drainedthrough the frit. The resin was washed once with DMF (2.5-5.0 mL). Tothe reaction vessel was added piperidine:DMF (20:80 v/v, 2.5-5.0 mL).The mixture was periodically agitated for 5 minutes and then thesolution was drained through the frit. The resin was washed 6 times asfollows: for each wash, DMF (2.5-5.0 mL) was added through the bottom ofthe vessel and the resulting mixture was periodically agitated for 30seconds before the solution was drained through the frit. To thereaction vessel was added NMM (0.8M in DMF, 3.0 mL, 48 eq) followed byChloroacetic anhydride (0.4M in DMF, 3.0 mL, 24 eq). The mixture wasperiodically agitated for 30 minutes, then the reaction solution wasdrained through the frit. The resin was washed once with DMF (4.0 mL).To the reaction vessel was added NMM (0.8M in DMF, 3.0 mL, 48 eq)followed by Chloroacetic anhydride (0.4M in DMF, 3.0 mL, 24 eq). Themixture was periodically agitated for 30 minutes, then the reactionsolution was drained through the frit. The resin was washed six timeswith DMF (3.0 mL) with periodically agitation of the mixture for 30seconds before the solution was drained through the frit. The resin wasthen washed five times with DCM (3.0 mL) with periodic agitation of theresulting mixture for 30 seconds before the solution was drained throughthe frit. The resulting resin was then dried with a stream of Nitrogenfor 10 mins.

Symphony Method B:

All manipulations were performed under automation on a Symphony peptidesynthesizer (Protein Technologies). All procedures unless noted wereperformed in a Symphony 20 mL polypropylene tube fitted with a bottomfrit. The tube connects to the Symphony peptide synthesizer through boththe bottom and the top of the tube. All Solvents, DMF, DCM, amino acidsand reagents are added through the bottom of the tube and pass upthrough the frit to contact the resin. All solutions are removed throughthe bottom of the tube. “Periodic agitation” describes a brief pulse ofN2 gas through the bottom frit; the pulse lasts approximately 5 secondsand occurs every 15 seconds. Amino acid solutions were generally notused beyond three weeks from preparation. HATU solution was used within5 days of preparation. DMF=dimethylformamide;HCTU=2-(6-Chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium;HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate; NMM=N-methyl morpholine;DIPEA=diisopropylethylamine; Sieber=Fmoc-amino-xanthen-3-yloxy, where“3-yloxy” describes the position and type of connectivity to thepolystyrene resin. The resin used is Merrifield polymer (polystyrene)with a Sieber linker (Fmoc-protected at nitrogen); 100-200 mesh, 1% DVB,0.71 mmol/g loading. Other common acid sensitive resins can also be usedin the synthesis such as Rink or functionalized Chloro trityl Resin.Common amino acids used are listed below with side-chain protectinggroups indicated inside parenthesis. Fmoc-Ala-OH; Fmoc-Arg(Pbf)-OH;Fmoc-Asn(Trt)-OH; Fmoc-Asp(OtBu)-OH; Fmoc-Bzt-OH; Fmoc-Cys(Trt)-OH;Fmoc-Dab(Boc)-OH; Fmoc-Dap(Boc)-OH; Fmoc-Gln(Trt)-OH; Fmoc-Gly-OH;Fmoc-His(Trt)-OH; Fmoc-t-Hyp(tBu)-OH (t-Hyp=trans-4-hydroxyproline;Fmoc-Ile-OH; Fmoc-Leu-OH; Fmoc-Lys(Boc)-OH; Fmoc-Nle-OH; Fmoc-Met-OH;Fmoc[N-Me]Ala-OH; Fmoc-[N-Me]Nle-OH; Fmoc-Phe-OH; Fmoc-Pro-OH;Fmoc-Sar-OH; Fmoc-Ser(tBu)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Trp(Boc)-OH;Fmoc-Tyr(tBu)-OH; Fmoc-Val-OH.

The procedures of “Symphony Method B” describes an experiment performedon a 0.10 mmol scale, where the scale is determined by the amount ofSieber linker bound to the resin. This scale corresponds toapproximately 140 mg of the Sieber-Merrifield resin described above. Allprocedures can be scaled beyond 0.10 mmol scale by adjusting thedescribed volumes by the multiple of the scale. Prior to amino acidcoupling, all peptide synthesis sequences began with a resin-swellingprocedure, described below as “Swelling procedure”. Coupling of aminoacids to a primary amine N-terminus used the “Standard-couplingprocedure” described below. Coupling of amino acids to a secondary amineN-terminus used the “Secondary amine-coupling procedure B”, Custom aminoacids are coupled via a manual blank addition of the amino acid “Customamino acids-coupling procedure” described below, and ChloroacetylAnhydride is added to the final position of the sequence using the“Final capping procedure” described below.

Swelling Procedure:

To a Symphony polypropylene reaction vessel was added Merrifield Sieberresin (140 mg, 0.100 mmol). The resin was washed (swelled) three timesas follows: to the reaction vessel was added DMF (2.5-5.0 mL) upon whichthe mixture was periodically agitated with N₂ bubbling from the bottomof the reaction vessel for 10 minutes before the solvent was drainedthrough the frit.

Standard-Coupling Procedure:

The resin was washed three times as follows: to the reaction vessel wasadded DMF (2.5 mL) upon which the mixture was periodically agitated withN₂ bubbling from the bottom of the reaction vessel for 30 seconds beforethe solvent was drained through the frit. To the reaction vessel wasadded piperidine:DMF (20:80 v/v, 5 mL). The mixture was periodicallyagitated for 5 minutes and then the solution was drained through thefrit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 5 mL).The mixture was periodically agitated for 5 minutes and then thesolution was drained through the frit. The resin was washed 6 times asfollows: for each wash, DMF (2.5 mL) was added through the bottom of thevessel and the resulting mixture was periodically agitated for 30seconds before the solution was drained through the frit. To thereaction vessel was added the amino acid (0.2M in DMF, 2.5 mL, 10 eq),then HATU (0.2M in DMF, 2.5 mL, 10 eq), and finally NMM (0.8M in DMF,2.5 mL, 20 eq). The mixture was periodically agitated for 30 minutes,then the reaction solution was drained through the frit. The resin waswashed 6 times as follows: DMF (2.5 mL) was added through the bottom ofthe vessel and the resulting mixture was periodically agitated for 30seconds before the solution was drained through the frit. To thereaction vessel was added Ac₂O/DIPEA/DMF (v/v/v 1:1:3 2.5 mL) themixture was periodically agitated for 10 minutes, then the reactionsolution was drained through the frit. The resin was washed successivelysix times as follows: for each wash, DMF (2.5 mL) was added through thebottom of the vessel and the resulting mixture was periodically agitatedfor 90 seconds before the solution was drained through the frit. Theresulting resin was used directly in the next step.

Secondary Amine-Coupling Procedure:

The resin was washed three times as follows: to the reaction vessel wasadded DMF (2.5 mL) upon which the mixture was periodically agitated withN2 bubbling from the bottom of the reaction vessel for 30 seconds beforethe solvent was drained through the frit. To the reaction vessel wasadded piperidine:DMF (20:80 v/v, 5 mL). The mixture was periodicallyagitated for 5 minutes and then the solution was drained through thefrit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 5 mL).The mixture was periodically agitated for 5 minutes and then thesolution was drained through the frit. The resin was washed 6 times asfollows: for each wash, DMF (2.5 mL) was added through the bottom of thevessel and the resulting mixture was periodically agitated for 30seconds before the solution was drained through the frit. To thereaction vessel was added the amino acid (0.2M in DMF, 2.5 mL, 10 eq),then HATU (0.2M in DMF, 2.5 mL, 10 eq), and finally NMM (0.8M in DMF,2.5 mL, 20 eq). The mixture was periodically agitated for 60 minutes,then the reaction solution was drained through the frit. The resin waswashed with DMF (6.25 mL) was added through the bottom of the vessel andthe resulting mixture was periodically agitated for 30 seconds beforethe solution was drained through the frit. To the reaction vessel wasadded the amino acid (0.2M in DMF, 2.5 mL, 10 eq.), then HATU (0.2M inDMF, 2.5 mL, 10 eq.), and finally NMM (0.8M in DMF, 2.5 mL, 20 eq.). Themixture was periodically agitated for 60 minutes, then the reactionsolution was drained through the frit. The resin was washed successivelythree times as follows: for each wash, DMF (2.5 mL) was added throughthe bottom of the vessel and the resulting mixture was periodicallyagitated for 30 seconds before the solution was drained through thefrit. To the reaction vessel was added Ac₂O/DIPEA/DMF (v/v/v 1:1:3 2.5mL) the mixture was periodically agitated for 10 minutes, then thereaction solution was drained through the frit. The resin was washedsuccessively six times as follows: for each wash, DMF (2.5 mL) was addedthrough the bottom of the vessel and the resulting mixture wasperiodically agitated for 90 seconds before the solution was drainedthrough the frit. The resulting resin was used directly in the nextstep.

Custom Amino Acids-Coupling Procedure:

The resin was washed three times as follows: to the reaction vessel wasadded DMF (2.5 mL) upon which the mixture was periodically agitated withN₂ bubbling from the bottom of the reaction vessel for 30 seconds beforethe solvent was drained through the frit. To the reaction vessel wasadded the amino acid (0.2M in DMF, 2.5 mL, 10 eq), then HATU (0.2M inDMF, 2.5 mL, 10 eq), and finally NMM (0.8M in DMF, 2.5 mL, 20 eq). Themixture was periodically agitated for six hours, then the reactionsolution was drained through the frit. The resin was washed 6 times asfollows: DMF (2.5 mL) was added through the bottom of the vessel and theresulting mixture was periodically agitated for 30 seconds before thesolution was drained through the frit. To the reaction vessel was addedAc₂O/DIPEA/DMF (v/v/v 1:1:3 2.5 mL) the mixture was periodicallyagitated for 10 minutes, then the reaction solution was drained throughthe frit. The resin was washed successively six times as follows: foreach wash, DMF (2.5 mL) was added through the bottom of the vessel andthe resulting mixture was periodically agitated for 90 seconds beforethe solution was drained through the frit. The resulting resin was useddirectly in the next step.

Final Capping Procedure:

The resin was washed three times as follows: to the reaction vessel wasadded DMF (2.5 mL) upon which the mixture was periodically agitated withN₂ bubbling from the bottom of the reaction vessel for 30 seconds beforethe solvent was drained through the frit. To the reaction vessel wasadded piperidine:DMF (20:80 v/v, 2.5-5.0 mL). The mixture wasperiodically agitated for 5 minutes and then the solution was drainedthrough the frit. The resin was washed once with DMF (2.5-5.0 mL). Tothe reaction vessel was added piperidine:DMF (20:80 v/v, 2.5-5.0 mL).The mixture was periodically agitated for 5 minutes and then thesolution was drained through the frit. The resin was washed six times asfollows: for each wash, DMF (2.5 mL) was added through the bottom of thevessel and the resulting mixture was periodically agitated for 30seconds before the solution was drained through the frit. To thereaction vessel was added NMM (0.8M in DMF, 2.5 mL, 20 eq) followed bythe addition of the Chloroacetic anhydride (0.4M in DMF, 2.5 mL, 10 eq).The mixture was periodically agitated for 15 minutes, then the reactionsolution was drained through the frit. The resin was washed with DMF(6.25 mL) was added through the bottom of the vessel and the resultingmixture was periodically agitated for 30 seconds before the solution wasdrained through the frit. To the reaction vessel was added NMM (0.8M inDMF, 2.5 mL, 10 eq) followed by the addition of the Chloroaceticanhydride (0.4M in DMF, 2.5 mL, 10 eq). The mixture was periodicallyagitated for 15 minutes, then the reaction solution was drained throughthe frit. The resin was washed 6 times as follows: DMF (2.5 mL) wasadded through the bottom of the vessel and the resulting mixture wasperiodically agitated for 30 seconds before the solution was drainedthrough the frit. To the reaction vessel was added Ac₂O/DIPEA/DMF (v/v/v1:1:3 2.5 mL) the mixture was periodically agitated for 10 minutes, thenthe reaction solution was drained through the frit. The resin was washedsuccessively six times as follows: for each wash, DMF (2.5 mL) was addedthrough the bottom of the vessel and the resulting mixture wasperiodically agitated for 30 seconds before the solution was drainedthrough the frit. The resin was washed successively four times asfollows: for each wash, DCM (2.5 mL) was added through the bottom of thevessel and the resulting mixture was periodically agitated for 30seconds before the solution was drained through the frit. The resultingresin was then dried with a stream of Nitrogen for 10 mins.

Manual Procedures Bromoacetic Acid Coupling:

All manipulations were performed manually at room temperature, unlessnoted otherwise. The resin was washed three times as follows: the resinsfrom four 0.100 mmol scale Symphony syntheses used to prepare ResinIntermediates A and B were combined into a 50 mL fritted glass reactorequipped with a three-way stopcock and washed with DMF (10 mL) threetimes with agitation by N₂ bubbling from the bottom of the reactionvessel. To the reaction vessel was added piperidine:DMF (20:80 v/v, 10mL). The mixture was periodically agitated for 5 minutes and then thesolution was drained through the frit. The procedure was repeated oncemore. The resin was washed six times with DMF (10 mL). To the reactionvessel was added a solution of bromoacetic acid (10.0 eq) in DMF (5.0mL) and DIC (10.3 eq). The mixture was agitated by N₂ bubbling for onehour, then the reaction solution was drained through the frit. The resinwas washed with DMF (5 mL) five times. The resulting resin was suspendedin DCM/DMF (3:2) and divided, by volume, into seven equal parts (0.057mmol), which were transferred into 25 mL fritted syringes. Each resinaliquot was used directly in the next on-resin N-alkylation step.

N-Gly-Alkylation On-Resin Method A:

All manipulations were performed manually at room temperature, unlessnoted otherwise. To the resin from the bromoacetylation step (0.057mmol) was added a solution of the required amine (10 eq.) and DIEA (11eq.) in DMF (mL) and the resulting mixture was stirred for one hour. Theresin was washed five times with DMF (10 mL). The procedure was repeatedonce more. When the hydrochloride salt of the amine was used, anadditional 10 eq. of DIEA was used. Reaction progress was monitored byTFA micro-cleavage of small resin samples. Upon reaction completion, theN-alkylated resin was washed five times with DMF (10 mL) and placed backinto a Symphony reaction vessel for completion of sequence assembly onthe Symphony peptide synthesizer.

N-Gly-Alkylation On-Resin Method B:

All manipulations were performed manually at room temperature, unlessnoted otherwise. To the resin from the bromoacetylation step (0.057mmol) was added a solution of the required amine (10 eq.) and DIEA (11eq.) in DMF (mL) and the resulting mixture was stirred for one hour. Theresin was washed five times with DMF (10 mL). The procedure was repeatedonce more. When the hydrochloride salt of the amine was used, anadditional 10 eq. of DIEA was used. The resin was then treated with asolution of amine (10-20 eq.) and DMAP (21 eq.) for one to sixteenhours. Reaction progress was monitored by TFA micro-cleavage of smallresin samples. Upon reaction completion, the N-alkylated resin waswashed five times with DMF (10 mL) and placed back into a Symphonyreaction vessel for completion of sequence assembly on the Symphonypeptide synthesizer.

N-Gly-Alkylation On-Resin Method C:

All manipulations were performed manually at room temperature, unlessnoted otherwise. This procedure was used when the required amine wasethylamine. To the resin from the bromoacetylation step (0.057 mmol) wasadded a solution of the ethylamine hydrochloride (10 eq.) and DIEA (21eq.) in DMF (mL) and the resulting mixture was stirred for one hour. Theresin was washed five times with DMF (10 mL). The procedure was repeatedonce more. The resin was then treated with a 2 M solution of ethylaminein THF (10 mL) for sixteen hours. Reaction progress was monitored by TFAmicro-cleavage of small resin samples. Upon reaction completion, theN-alkylated resin was washed five times with DMF (10 mL) and placed backinto a Symphony reaction vessel for completion of sequence assembly onthe Symphony peptide synthesizer.

N-Gly-Alkylation On-Resin Method D

All manipulations were performed manually at room temperature, unlessnoted otherwise. To the bromoacetylated resin (0.100 mmol) was added asolution of the required amine (10 eq., 1.0 mmol) and DBU (5 eq., 0.5mmol) in DMF (3 mL) and the resulting mixture was stirred for threehours. The resin was washed once with DMF (5 mL). The procedure wasrepeated once more, but the reaction was allowed to proceed for 16 hrs.The resin was washed four times with DMF (4 mL) and DCM (4 mL), and wasthen placed back into a Symphony reaction vessel for completion ofsequence assembly on the Symphony peptide synthesizer.

Alkylation Method A:

A solution of the alcohol corresponding to the alkylating group (0.046g, 1.000 mmol), triphenylphosphine (0.131 g, 0.500 mmol), and DIAD(0.097 mL, 0.500 mmol) in 3 mL of THF was added to nosylated resin(0.186 g, 0.100 mmol), and the reaction mixture was stirred for 16 hoursat room temperature. The resin was washed three times with THF (5 mL)Tetrahydrofuran, and the above procedure was repeated 1-3 times.Reaction progress was monitored by TFA micro-cleavage of small resinsamples treated with a solution of 50 μL of TIS in 1 mL of TFA for 1.5hours.

Alkylation Method B:

The nosylated resin (0.100 mmol) was washed three times withN-methylpyrrolidone (NMP) (3 mL). A solution of NMP (3 mL), AlkylBromide (20 eq, 2.000 mmol) and DBU (20 eq, 0.301 mL, 2.000 mmol) wasadded to the resin, and the reaction mixture was stirred for 16 hours atroom temperature. The resin was washed with NMP (3 mL) and the aboveprocedure was repeated once more. Reaction progress was monitored by TFAmicro-cleavage of small resin samples treated with a solution of 50 μLof TIS in 1 mL of TFA for 1.5 hours.

Nosylate Formation:

A solution of collidine (10 eq.) in DCM (2 mL) was added to the resin,followed by a solution of Nos-Cl (8 eq.) in DCM (1 mL). The reactionmixture was stirred for 16 hours at room temperature. The resin waswashed three times with DCM (4 mL) and three times with DMF (4 mL). Thealternating DCM and DMF washes were repeated three times, followed byone final set of four DCM washes (4 mL).

Nosylate Removal:

The resin (0.100 mmol) was swelled using three washes with DMF (3 mL)and three washes with NMP (3 mL). A solution of NMP (3 mL), DBU (0.075mL, 0.500 mmol) and 2-mercaptoethanol (0.071 mL, 1.000 mmol) was addedto the resin and the reaction mixture was stirred for 5 minutes at roomtemperature. After filtering and washing with NMP (3 mL), the resin wasre-treated with a solution of NMP (3 mL), DBU (0.075 mL, 0.500 mmol) and2-mercaptoethanol (0.071 mL, 1.000 mmol) for 5 minutes at roomtemperature. The resin was washed three times with NMP (3 mL), fourtimes with DMF (4 mL) and four times with DCM (4 mL), and was placedback into a Symphony reaction vessel for completion of sequence assemblyon the Symphony peptide synthesizer.

Chloroacetyl Chloride Coupling Procedure:

To the reaction vessel containing the resin from the previous step wasadded piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodicallyagitated for 5 minutes and then the solution was drained through thefrit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 5.0mL). The mixture was periodically agitated for 5 minutes and then thesolution was drained through the frit. The resin was washed successivelysix times as follows: for each wash, DMF (5.0 mL) was added through thetop of the vessel and the resulting mixture was periodically agitatedfor 30 seconds before the solution was drained through the frit. To thereaction vessel was added 3.5 mL of a solution of DIPEA (40 eq), andchloroacetyl chloride (20 eq) in DMF. The mixture was periodicallyagitated for three hours, then the solution was drained through thefrit. The resin was washed successively five times as follows: for eachwash, DMF (5.0 mL) was added to top of the vessel and the resultingmixture was periodically agitated for 60 seconds before the solution wasdrained through the frit. The resin was washed successively three timesas follows: for each wash, CH₂C₁₂ (5.0 mL) was added to top of thevessel and the resulting mixture was periodically agitated for 60seconds before the solution was drained through the frit. The resin wasthen dried under high vacuum.

CEM Method A:

All manipulations were performed under automation on a CEM Libertymicrowave peptide synthesizer (CEM Corporation). All procedures unlessnoted were performed in a 30 or 125 mL polypropylene tube fitted with abottom frit to a CEM Discovery microwave unit. The tube connects to theCEM Liberty synthesizer through both the bottom and the top of the tube.DMF and DCM can be added through the top and bottom of the tube, whichwashes down the sides of the tube equally. All solutions are removedthrough the bottom of the tube except while transferring resin from thetop. “Periodic bubbling” describes a brief bubbling of N2 gas throughthe bottom frit. Amino acid solutions were generally not used beyondthree weeks from preparation. HATU solution was used within 5 days ofpreparation. DMF=dimethylformamide;HCTU=2-(6-Chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium;HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate; DIPEA=diisopropylethylamine;Sieber=Fmoc-amino-xanthen-3-yloxy, where “3-yloxy” describes theposition and type of connectivity to the polystyrene resin. The resinused is Merrifield polymer (polystyrene) with a Sieber linker(Fmoc-protected at nitrogen); 100-200 mesh, 1% DVB, 0.71 mmol/g loading.Common amino acids used are listed below with side-chain protectinggroups indicated inside parenthesis. Fmoc-Ala-OH; Fmoc-Arg(Pbf)-OH;Fmoc-Asn(Trt)-OH; Fmoc-Asp(OtBu)-OH; Fmoc-Bzt-OH; Fmoc-Cys(Trt)-OH;Fmoc-Dab(Boc)-OH; Fmoc-Dap(Boc)-OH; Fmoc-Gln(Trt)-OH; Fmoc-Gly-OH;Fmoc-His(Trt)-OH; Fmoc-Hyp(tBu)-OH; Fmoc-Ile-OH; Fmoc-Leu-OH;Fmoc-Lys(Boc)-OH; Fmoc-Nle-OH; Fmoc-Met-OH; Fmoc-[N-Me]Ala-OH;Fmoc[N-Me]Nle-OH; Fmoc-Phe-OH; Fmoc-Pro-OH; Fmoc-Sar-OH;Fmoc-Ser(tBu)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Trp(Boc)-OH; Fmoc-Tyr(tBu)-OH;Fmoc-Val-OH

The procedures of “CEM Method A” describe an experiment performed on a0.100 mmol scale, where the scale is determined by the amount of Sieberlinker bound to the resin. This scale corresponds to approximately 140mg of the Sieber-Merrifield resin described above. All procedures can bescaled beyond 0.100 mmol scale by adjusting the described volumes by themultiple of the scale. Prior to amino acid coupling, all peptidesynthesis sequences began with a resin-swelling procedure, describedbelow as “Resin-swelling procedure”. Coupling of amino acids to aprimary amine N-terminus used the “Single-coupling procedure” describedbelow. Coupling of amino acids to a secondary amine N-terminus used the“Secondary amine-coupling procedure” described below. Coupling ofchloroacetyl group to the N-terminus of the peptide is described by the“Chloroacetyl chloride coupling procedure” or “Chloroacetic acidcoupling procedure” detailed above.

Resin-Swelling Procedure:

To 50 mL polypropylene conical tube was added Merrifield: Sieber resin(140 mg, 0.100 mmol). Then DMF (7 mL) was added to the tube followed byDCM (7 mL). The resin was then transferred to the reaction vessel fromtop of the vessel. The procedure is repeated additionally two times. DMF(7 mL) was added followed by DCM (7 mL). The resin was allowed to swellwith N2 bubbling from the bottom of the reaction vessel for 15 minutesbefore the solvent was drained through the frit.

Standard Coupling Procedure:

To the reaction vessel containing resin from the previous step was addeda solution of piperidine:DMF (20:80 v/v, 5.0 mL). The mixture wasperiodically agitated for 3 minutes and then the solution was drainedthrough the frit. To the reaction vessel was added a solution ofpiperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodicallyagitated for 3 minutes and then the solution was drained through thefrit. The resin was washed successively three times as follows: DMF (7mL) wash from top, followed by DMF (7 mL) wash from bottom and finallywith DMF (7 mL) wash from top. To the reaction vessel was added theamino acid (0.2M in DMF, 2.5 mL, 5 eq), HATU (0.5M in DMF, 1.0 mL, 5eq), and DIPEA (2M in NMP, 0.5 mL, 10 eq). The mixture was mixed by N2bubbling for 5 minutes at 75° C. for all amino acids, exceptFmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH which are coupled at 50° C., thereaction solution was drained through the frit. The resin was washedsuccessively three times as follows: DMF (7 mL) wash from top, followedby DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash fromtop. To the reaction vessel was added a solution of aceticanhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). The mixture was periodicallybubbled for 2 minutes at 65° C., then the solution was drained throughthe frit. The resin was washed successively three times as follows: DMF(7 mL) wash from top, followed by DMF (7 mL) wash from bottom andfinally with DMF (7 mL) wash from top. The resulting resin was useddirectly in the next step.

Double-Couple Coupling Procedure:

To the reaction vessel containing resin from the previous step was addeda solution of piperidine:DMF (20:80 v/v, 5.0 mL). The mixture wasperiodically agitated for 3 minutes and then the solution was drainedthrough the frit. To the reaction vessel was added a solution ofpiperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodicallyagitated for 3 minutes and then the solution was drained through thefrit. The resin was washed successively three times as follows: DMF (7mL) wash from top, followed by DMF (7 mL) wash from bottom and finallywith DMF (7 mL) wash from top. To the reaction vessel was added theamino acid (0.2M in DMF, 2.5 mL, 5 eq), HATU (0.5M in DMF, 1.0 mL, 5eq), and DIPEA (2M in NMP, 0.5 mL, 10 eq). The mixture was mixed by N2bubbling for 5 minutes at 75° C. for all amino acids, exceptFmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH which are coupled at 50° C., thereaction solution was drained through the frit. The resin was washedsuccessively three times as follows: DMF (7 mL) wash from top, followedby DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash fromtop. To the reaction vessel was added the amino acid (0.2M in DMF, 2.5mL, 5 eq), HATU (0.5M in DMF, 1.0 mL, 5 eq), and DIPEA (2M in NMP, 0.5mL, 10 eq). The mixture was mixed by N2 bubbling for 5 minutes at 75° C.for all amino acids, except Fmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH whichare coupled at 50° C., the reaction solution was drained through thefrit. The resin was washed successively three times as follows: DMF (7mL) wash from top, followed by DMF (7 mL) wash from bottom and finallywith DMF (7 mL) wash from top. To the reaction vessel was added asolution of acetic anhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). Themixture was periodically bubbled for 2 minutes at 65° C., then thesolution was drained through the frit. The resin was washed successivelythree times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL)wash from bottom and finally with DMF (7 mL) wash from top. Theresulting resin was used directly in the next step.

Custom Amino Acids-Coupling Procedure:

To the reaction vessel containing resin from the previous step was addeda solution of piperidine:DMF (20:80 v/v, 5.0 mL). The mixture wasperiodically agitated for 3 minutes and then the solution was drainedthrough the frit. To the reaction vessel was added a solution ofpiperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodicallyagitated for 3 minutes and then the solution was drained through thefrit. The resin was washed successively three times as follows: DMF (7mL) wash from top, followed by DMF (7 mL) wash from bottom and finallywith DMF (7 mL) wash from top. To the reaction vessel was added theamino acid solution (1.25 mL to 5 mL, 2.5 eq to 10 eq) containing HATU(2.5 eq to 10 eq), and finally DIPEA (2M in NMP, 0.5 mL to 1 mL, 20 eq).The mixture was mixed by N2 bubbling for 5 minutes to 2 hours at 25° C.to 75° C., then the reaction solution was drained through the frit. Theresin was washed successively three times as follows: DMF (7 mL) washfrom top, followed by DMF (7 mL) wash from bottom and finally with DMF(7 mL) wash from top. To the reaction vessel was added a solution ofacetic anhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). The mixture wasperiodically bubbled for 2 minutes at 65° C., then the solution wasdrained through the frit. The resin was washed successively three timesas follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash frombottom and finally with DMF (7 mL) wash from top. The resulting resinwas used directly in the next step.

Chloroacetyl Chloride Coupling Procedure:

To the reaction vessel containing the resin from the previous step wasadded piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodicallyagitated for 3 minutes and then the solution was drained through thefrit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 5.0mL). The mixture was periodically agitated for 3 minutes and then thesolution was drained through the frit. The resin was washed successivelyfive times as follows: for each wash, DMF (4.0 mL) was added through thetop of the vessel and the resulting mixture was periodically agitatedfor 30 seconds before the solution was drained through the frit. To thereaction vessel was added 3.0 mL of a solution of DIPEA (4.0 mmol, 0.699mL, 40 eq), and chloroacetyl chloride (2.0 mmol, 0.160 mL, 20 eq) inDMF. The mixture was periodically agitated for 12 to 18 hours, then thesolution was drained through the frit. The resin was washed successivelythree times as follows: for each wash, DMF (4.0 mL) was added to top ofthe vessel and the resulting mixture was periodically agitated for 90seconds before the solution was drained through the frit. The resin waswashed successively four times as follows: for each wash, CH₂C₁₂ (2.0mL) was added to top of the vessel and the resulting mixture wasperiodically agitated for 90 seconds before the solution was drainedthrough the frit.

Global Deprotection Method A:

All manipulations were performed manually at room temperature unlessnoted otherwise. The procedure of “Global Deprotection Method A”describes an experiment performed on a 0.057-0.100 mmol scale, where thescale is determined by the amount of Rink or Sieber linker bound to theresin. The procedure can be scaled beyond 0.057-0.100 mmol scale byadjusting the described volumes by the multiple of the scale. A“deprotection solution” was prepared using trifluoroaceticacid:triisopropylsilane:dithiothreitol (95:2.5:2.5 v:v:w) ortrifluoroacetic acid:triisopropylsilane:dithiothreitol (96.5:2.5:1.0v:v:w). The solution was pre-cooled in ice prior to adding it to theresin. To the resin in a 25 mL fritted syringe was added the“deprotection solution” (2.5-4.0 mL). The mixture was mixed in a shakerfor 60 min. The solution was filtered through the frit into cold diethylether (30 mL). The precipitated solid was centrifuged for 3 min. Thesupernatant solution was decanted and the solid was re-suspended indiethyl ether (15 mL). This procedure was repeated two more times. Thesupernatant was decanted and the remaining solid was dried under highvacuum. The crude peptide was obtained as a white to off-white solid.

Cyclization Method A:

All manipulations were performed manually unless noted otherwise. Theprocedure of “Cyclization Method A” describes an experiment performed ona 0.057-0.100 mmol scale. The crude peptide solid was dissolved in asolution of acetonitrile:aqueous 0.1M ammonium bicarbonate buffer (1:3or 1:2, v:v; 30-40 mL), and the pH of the solution was carefullyadjusted to 8.5-9.0 using aqueous NaOH (1.0 M). The solution was thenmixed using a shaker for 12 to 18 hours. The reaction solution wasconcentrated and the residue was then dissolved in acetonitrile:water.This solution was subjected to reverse-phase HPLC purification to affordthe desired cyclic peptide.

Preparation of Intermediate Resins for Examples 3003-3050 Preparation ofIntermediate Resin A:

To a Symphony 20 mL reaction vessels was added Sieber resin (0.100 mmol)and the vessel was placed on the Symphony peptide synthesizer. Thefollowing procedures were then performed sequentially:

Fmoc-Gly-OH “Symphony Method B: Resin-swelling procedure” was followed;Fmoc-Cys(Trt)-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-Leu-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-Ala-OH “Symphony Method B: Standard-coupling procedure” wasfollowed. The Fmoc group was removed using 20% Piperidine/DMF (5 mL) for5 mins with periodic Nitrogen stirring. The resin was washed with DMF(2.5 mL) and then 20% Piperidine/DMF (5 mL) was added to the resin, andperiodic Nitrogen stirring was continued for 5 mins. The resulting resinwas washed six times with DMF (2.5 mL), then five times with DCM (2.5mL), and was used as an intermediate for the N-alkylation procedures.

Preparation of Intermediate Resin B:

To a Symphony 20 mL reaction vessels was added Sieber resin (0.100 mmol)and the vessel was placed on the Symphony peptide synthesizer. Thefollowing procedures were then performed sequentially:

Fmoc-Gly-OH “Symphony Method B: Resin-swelling procedure” was followed;Fmoc-Cys(Trt)-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-Leu-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-Nle-OH “Symphony Method B: Standard-coupling procedure” wasfollowed. The Fmoc group was removed using 20% Piperidine/DMF (5 mL) for5 mins with periodic Nitrogen stirring. The resin was washed with DMF(2.5 mL) and then 20% Piperidine/DMF (5 mL) was added to the resin, andperiodic Nitrogen stirring was continued for 5 mins. The resulting resinwas washed six times with DMF (2.5 mL), then five times with DCM (2.5mL), and was used as an intermediate for the N-alkylation procedures.

Preparation of Intermediate Resin C:

To a Symphony 20 mL reaction vessels was added Sieber resin (0.100 mmol)and the vessel was placed on the Symphony peptide synthesizer. Thefollowing procedures were then performed sequentially:

Fmoc-Gly-OH “Symphony Method B: Resin-swelling procedure” was followed;Fmoc-Cys(Trt)-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-Leu-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-nMethyl-Nle-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-Nle-OH “Symphony Method B: Secondary amine-coupling procedure” wasfollowed.

The Fmoc group was removed using 20% Piperidine/DMF (5 mL) for 5 minswith periodic Nitrogen stirring. The resin was washed with DMF (2.5 mL)and then 20% Piperidine/DMF (5 mL) was added to the resin, and periodicNitrogen stirring was continued for 5 mins. The resulting resin waswashed six times with DMF (2.5 mL), then five times with DCM (2.5 mL),and was used as an intermediate for the N-alkylation procedures.

Preparation of Intermediate Resin D:

To a Symphony 20 mL reaction vessels was added Sieber resin (0.100 mmol)and the vessel was placed on the Symphony peptide synthesizer. Thefollowing procedures were then performed sequentially:

Fmoc-Gly-OH “Symphony Method B: Resin-swelling procedure” was followed;Fmoc-Cys(Trt)-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-Leu-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-nMethyl-Nle-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-Ala-OH “Symphony Method B: Secondary amine-coupling procedure” wasfollowed.

The Fmoc group was removed using 20% Piperidine/DMF (5 mL) for 5 minswith periodic Nitrogen stirring. The resin was washed with DMF (2.5 mL)and then 20% Piperidine/DMF (5 mL) was added to the resin, and periodicNitrogen stirring was continued for 5 mins. The resulting resin waswashed six times with DMF (2.5 mL), then five times with DCM (2.5 mL),and was used as an intermediate for the N-alkylation procedures.

Preparation of Intermediate Resin E:

To a Symphony 20 mL reaction vessels was added Sieber resin (0.100 mmol)and the vessel was placed on the Symphony peptide synthesizer. Thefollowing procedures were then performed sequentially:

Fmoc-Gly-OH “Symphony Method B: Resin-swelling procedure” was followed;Fmoc-Cys(Trt)-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-Leu-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-nMethyl-Nle-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-nMethyl-Nle-OH “Symphony Method B: Secondary amine-couplingprocedure”, was followed;Fmoc-Trp(Boc)-OH “Symphony Method B: Secondary amine-couplingprocedure”, was followed;Fmoc-Dab(Boc)-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-Trp(Boc)-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-Hyp(tBu)-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-Glu(tBu)-OH “Symphony Method B: Secondary amine-coupling procedure”was followed;Fmoc-His(Trt)-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-Pro-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-Asn(Trt)-OH “Symphony Method B: Secondary amine-coupling procedure”was followed;Fmoc-Ala-OH “Symphony Method B: Standard-coupling procedure” wasfollowed. The Fmoc group was removed using 20% Piperidine/DMF (5 mL) for5 mins with periodic Nitrogen stirring. The resin was washed with DMF(2.5 mL) and then 20% Piperidine/DMF (5 mL) was added to the resin, andperiodic Nitrogen stirring was continued for 5 mins. The resulting resinwas washed six times with DMF (2.5 mL), then five times with DCM (2.5mL), and was used as an intermediate for the N-alkylation procedures.

Preparation of Intermediate Resin F:

To a Symphony 20 mL reaction vessels was added Sieber resin (0.100 mmol)and the vessel was placed on the Symphony peptide synthesizer. Thefollowing procedures were then performed sequentially:

Fmoc-Gly-OH “Symphony Method B: Resin-swelling procedure” was followed;Fmoc-Cys(Trt)-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-Leu-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-nMethyl-Nle-OH “Symphony Method B: Standard-coupling procedure” wasfollowed.

The Fmoc group was removed using 20% Piperidine/DMF (5 mL) for 5 minswith periodic Nitrogen stirring. The resin was washed with DMF (2.5 mL)and then 20% Piperidine/DMF (5 mL) was added to the resin, and periodicNitrogen stirring was continued for 5 mins. The resulting resin waswashed six times with DMF (2.5 mL), and was then transferred to a BioRadtube, and treated with the following solution: Bromoacetic acid (10 eq.,1 mmol) and DIC (11 eq., 1.1 mmol) in DMF (3 mL). The reaction mixturewas stirred for 3 hours. After filtration and a DMF wash (3 ML), theresin was re-treated with the following solution: Bromoacetic acid (10eq., 1 mmol) and DIC (11 eq., 1.1 mmol) in DMF (3 mL). The reactionmixture was stirred for 16 hours. After filtration, the resin was washedfive times with DMF (3.0 mL), then five times with DCM (2.5 mL), and wasused as an intermediate for the N-alkylation procedures.

Preparation of Intermediate Resin G:

To a Symphony 20 mL reaction vessels was added Sieber resin (0.100 mmol)and the vessel was placed on the Symphony peptide synthesizer. Thefollowing procedures were then performed sequentially:

Fmoc-Gly-OH “Symphony Method B: Resin-swelling procedure” was followed;Fmoc-Cys(Trt)-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-Leu-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-nMethyl-Nle-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-nMethyl-Nle-OH “Symphony Method B: Secondary amine-couplingprocedure” was followed;Fmoc-Trp(Boc)-OH “Symphony Method B: Secondary amine-coupling procedure”was followed;Fmoc-Dab(Boc)-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-Trp(Boc)-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-Hyp(tBu)-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-Glu(tBu)-OH “Symphony Method B: Secondary amine-coupling procedure”was followed;Fmoc-His(Trt)-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-Pro-OH “Symphony Method B: Standard-coupling procedure” wasfollowed;Fmoc-Asn(Trt)-OH “Symphony Method B: Secondary amine-coupling procedure”was followed.

The Fmoc group was removed using 20% Piperidine/DMF (5 mL) for 5 minswith periodic Nitrogen stirring. The resin was washed with DMF (2.5 mL)and then 20% Piperidine/DMF (5 mL) was added to the resin, and periodicNitrogen stirring was continued for 5 mins. The resulting resin waswashed six times with DMF (2.5 mL), and was then transferred to a BioRadtube, and treated with the following solution: Bromoacetic acid (10 eq.,1 mmol) and DIC (11 eq., 1.1 mmol) in DMF (3 mL). The reaction mixturewas stirred for 3 hours. After filtration and a DMF wash (3 ML), theresin was re-treated with the following solution: Bromoacetic acid (10eq., 1 mmol) and DIC (11 eq., 1.1 mmol) in DMF (3 mL). The reactionmixture was stirred for 16 hours. After filtration, the resin was washedfive times with DMF (3.0 mL), then five times with DCM (2.5 mL), and wasused as an intermediate for the N-alkylation procedures.

Preparation of Intermediate Resin H.

To four Symphony reaction vessels was added Rink resin (mg, 0.100 mmol)and the vessels were placed on the Symphony peptide synthesizer. Thefollowing procedures were then performed sequentially:

“Symphony Method A: Resin-swelling procedure” was followed;“Symphony Method A: Standard coupling procedure” was followed withFmoc-Gly-OH;“Symphony Method A: Standard coupling procedure” was followed withFmoc-Cys(Trt)-OH;“Symphony Method A: Standard coupling procedure” was followed withFmoc-Leu-OH;“Symphony Method A: Secondary-amine Coupling procedure” was followedwith Fmoc-[N-Me]Nle-OH;“Symphony Method A: Secondary-amine Coupling procedure” was followedwith Fmoc-[N-Me]Nle-OH“Symphony Method A: Secondary-amine Coupling procedure” was followedwith Fmoc-Trp(Boc)-OH;“Symphony Method A: Standard coupling procedure” was followed withFmoc-Dab(Boc)-OH;“Symphony Method A: Standard coupling procedure” was followed withFmoc-Trp(Boc)-OH.The resulting resins were combined into a 50 mL fritted glass reactorand washed with DMF as described in the above bromoacetic acid couplingprocedure.

Preparation of Intermediate Resin I.

To four Symphony reaction vessels was added Rink resin (mg, 0.100 mmol)and the vessels were placed on the Symphony peptide synthesizer. Thefollowing procedures were then performed sequentially:

“Symphony Method A: Resin-swelling procedure” was followed;“Symphony Method A: Standard coupling procedure” was followed withFmoc-Gly-OH;“Symphony Method A: Standard coupling procedure” was followed withFmoc-Cys(Trt)-OH;“Symphony Method A: Standard coupling procedure” was followed withFmoc-Leu-OH;“Symphony Method A: Secondary-amine coupling procedure” was followedwith Fmoc-[N-Me]Nle-OH;“Symphony Method A: Secondary-amine coupling procedure” was followedwith Fmoc-[N-Me]Nle-OH“Symphony Method A: Secondary-amine coupling procedure” was followedwith Fmoc-Trp(Boc)-OH;“Symphony Method A: Standard coupling procedure” was followed withFmoc-Dab(Boc)-OH;“Symphony Method A: Standard coupling procedure” was followed withFmoc-Trp(Boc)-OH.“Symphony Method A: Standard coupling procedure” was followed withFmoc-t-Hyp(tBu)-OH;“Symphony Method A: Secondary-amine Coupling procedure” was followedwith Fmoc-Glu(OtBu)-OH;“Symphony Method A: Standard coupling procedure” was followed withFmoc-His(Trt)-OH.The resulting resins were combined into a 50 mL fritted glass reactorand washed with DMF as described in the above bromoacetic acid couplingprocedure.

Preparation of Example 3003

Example 3003 was prepared following the general synthetic sequencedescribed below, starting from Intermediate Resin H. Intermediate ResinA (0.400 mmol) was placed into a 50 mL fritted glass reactor. Thefollowing procedures were then performed sequentially:

“Bromoacetic Acid Coupling” procedure was followed;“N-Gly-Alkylation On-resin Method C” procedure was followed;“Symphony Method A: Secondary-amine coupling without Fmoc deprotection”procedure was followed with Fmoc-Glu(OtBu)-OH;“Symphony Method A: Standard coupling procedure” was followed withFmoc-His(Trt)-OH;“Symphony Method A: Standard coupling procedure” was followed withFmoc-Pro-OH;“Symphony Method A: Secondary-amine coupling procedure” was followedwith Fmoc-Asn(Trt)-OH;Symphony Method A: Standard coupling procedure” was followed withFmoc-[N-Me]Ala-OH;“Symphony Method A: Secondary-amine coupling procedure” was followedwith Fmoc-Tyr(tBu)-OH;“Chloroacetic Anhydride capping procedure” was followed;“Global Deprotection Method A” was followed;“Cyclization Method A” was followed.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 45-85% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of product was 6.3 mg, and its estimated purityby LCMS analysis was 99% using “Analysis LCMS condition D”.

Analysis LCMS condition D: Retention time=1.88 min; ESI-MS(+) m/z 935.1(M+2H).

ESI-HRMS(+) m/z:

Calculated: 934.4692 (M+2H).

Found: 934.4674 (M+2H).

Preparation of Example 3004

Example 3004 was prepared following the general synthetic sequencedescribed for the preparation of Example 3003, starting fromIntermediate Resin H, using the following general procedures:“Bromoacetic Acid Coupling” procedure; “N-Gly-Alkylation On-resin MethodA” procedure; “Symphony Method A: Secondary-amine coupling without Fmocdeprotection” procedure; “Symphony Method A: Standard couplingprocedure”; “Symphony Method A: Secondary-amine coupling procedure”;“Chloroacetic Anhydride capping” procedure; “Global Deprotection MethodA”; “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 50-90% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of product was 6.6 mg, and its estimated purityby LCMS analysis was 95% using “Analysis LCMS condition D”.

Analysis LCMS condition D: Retention time=1.87 min; ESI-MS(+) m/z 961.9(M+2H).

ESI-HRMS(+) m/z:

Calculated: 965.4771 (M+2H).

Found: 965.4754 (M+2H).

Preparation of Example 3005

Example 3005 was prepared following the general synthetic sequencedescribed for the preparation of Example 3003, starting fromIntermediate Resin H, using the following general procedures:“Bromoacetic Acid Coupling” procedure; “N-Gly-Alkylation On-resin MethodB” procedure; “Symphony Method A: Secondary-amine coupling without Fmocdeprotection” procedure; “Symphony Method A: Standard couplingprocedure”; “Symphony Method A: Secondary-amine coupling procedure”;“Chloroacetic Anhydride capping” procedure; “Global Deprotection MethodA”; “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 45-85% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of product was 3.9 mg, and its estimated purityby LCMS analysis was 100% using “Analysis LCMS condition D”.

Analysis LCMS condition D: Retention time=1.72 min; ESI-MS(+) m/z 950.4(M+2H).

ESI-HRMS(+) m/z:

Calculated: 949.4563 (M+2H).

Found: 949.4547 (M+2H).

Preparation of Example 3006

Example 3006 was prepared following the general synthetic sequencedescribed for the preparation of Example 3003, starting fromIntermediate Resin H, using the following general procedures:“Bromoacetic Acid Coupling” procedure; “N-Gly-Alkylation On-resin MethodA” procedure; “Symphony Method A: Secondary-amine coupling without Fmocdeprotection” procedure; “Symphony Method A: Standard couplingprocedure”; “Symphony Method A: Secondary-amine coupling procedure”;“Chloroacetic Anhydride capping” procedure; “Global Deprotection MethodA”; “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 50-90% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of product was 13.8 mg, and its estimated purityby LCMS analysis was 100% using “Analysis LCMS conditions D and E”.

Analysis LCMS condition D: Retention time=1.90 min; ESI-MS(+) m/z 950.5(M+2H).

Analysis LCMS condition E: Retention time=1.58 min; ESI-MS(+) m/z 950.1

ESI-HRMS(+) m/z:

Calculated: 949.4745 (M+2H).

Found: 949.4725 (M+2H).

Preparation of Example 3007

Example 3007 was prepared following the general synthetic sequencedescribed for the preparation of Example 3003, starting fromIntermediate Resin H, using the following general procedures:“Bromoacetic Acid Coupling” procedure; “N-Gly-Alkylation On-resin MethodB” procedure; “Symphony Method A: Secondary-amine coupling without Fmocdeprotection” procedure; “Symphony Method A: Standard couplingprocedure”; “Symphony Method A: Secondary-amine coupling” procedure;“Chloroacetic Anhydride capping” procedure; “Global Deprotection MethodA”; “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 50-90% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of product was 3.9 mg, and its estimated purityby LCMS analysis was 100% using “Analysis LCMS condition D”.

Analysis LCMS condition D: Retention time=1.96 min; ESI-MS(+) m/z 969.3(M+2H).

ESI-HRMS(+) m/z:

Calculated: 968.4629 (M+2H).

Found: 968.4617 (M+2H).

Preparation of Example 3008

Example 3008 was prepared following the general synthetic sequencedescribed below, starting from Intermediate Resin I. Intermediate ResinB (0.400 mmol) was placed into a 50 mL fritted glass reactor. Thefollowing procedures were then performed sequentially:

“Bromoacetic Acid Coupling” procedure was followed;“N-Gly-Alkylation On-resin Method A” procedure was followed;“Symphony Method A: Secondary-amine coupling without Fmoc deprotection”procedure was followed with Fmoc-Asn(Trt)-OH;Symphony Method A: Standard coupling procedure” was followed withFmoc-[N-Me]Ala-OH;“Symphony Method A: Secondary-amine coupling procedure” was followedwith Fmoc-Tyr(tBu)-OH;“Chloroacetic Anhydride capping procedure” was followed;“Global Deprotection Method A” was followed;“Cyclization Method A” was followed.The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 50-90% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of product was 10.6 mg, and its estimated purityby LCMS analysis was 99% using “Analysis LCMS condition D”.

Analysis LCMS condition D: Retention time=1.87 min; ESI-MS(+) m/z 974.3(M+2H).

ESI-HRMS(+) m/z:

Calculated: 973.4745 (M+2H).

Found: 973.4729 (M+2H).

Preparation of Example 3009

Example 3009 was prepared following the general synthetic sequencedescribed for the preparation of Example 3008, starting fromIntermediate Resin I, using the following general procedures:“Bromoacetic Acid Coupling” procedure; “N-Gly-Alkylation On-resin MethodA” procedure; “Symphony Method A: Secondary-amine coupling without Fmocdeprotection” procedure; Symphony Method A: Standard coupling”procedure; “Symphony Method A: Secondary-amine coupling” procedure;“Chloroacetic Anhydride capping” procedure; “Global Deprotection MethodA”; “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 45-85% B over 35minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of product was 9.1 mg, and its estimated purityby LCMS analysis was 100% using “Analysis LCMS condition E”.

Analysis LCMS condition E: Retention time=1.56 min; ESI-MS(+) m/z 958.0(M+2H).

ESI-HRMS(+) m/z:

Calculated: 957.4720 (M+2H).

Found: 957.4693 (M+2H).

Preparation of Example 3010

Example 3010 was prepared following the general synthetic sequencedescribed for the preparation of Example 3008, starting fromIntermediate Resin I, using the following general procedures:“Bromoacetic Acid Coupling” procedure; “N-Gly-Alkylation On-resin MethodB” procedure; “Symphony Method A: Secondary-amine coupling without Fmocdeprotection” procedure; Symphony Method A: Standard coupling”procedure; “Symphony Method A: Secondary-amine coupling” procedure;“Chloroacetic Anhydride capping” procedure; “Global Deprotection MethodA”; “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 50-90% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of product was 4.7 mg, and its estimated purityby LCMS analysis was 97% using “Analysis LCMS condition D”.

Analysis LCMS condition D: Retention time=1.83 min; ESI-MS(+) m/z 977.7(M+2H).

ESI-HRMS(+) m/z:

Calculated: 976.4604 (M+2H).

Found: 976.4592 (M+2H).

Preparation of Example 3011

Example 3011 was prepared following the general synthetic sequencedescribed for the preparation of Example 3003, starting fromIntermediate Resin H, using the following general procedures:“Bromoacetic Acid Coupling” procedure; “N-Gly-Alkylation On-resin MethodA” procedure; “Symphony Method A: Secondary-amine coupling without Fmocdeprotection” procedure; “Symphony Method A: Standard couplingprocedure”; “Symphony Method A: Secondary-amine coupling procedure”;“Chloroacetic Anhydride capping” procedure; “Global Deprotection MethodA”; “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 50-90% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of product was 10.6 mg, and its estimated purityby LCMS analysis was 89% using “Analysis LCMS conditions D and E”.

Analysis LCMS condition D: Retention time=1.85 min; ESI-MS(+) m/z 942.9(M+2H).

Analysis LCMS condition E: Retention time=1.47 min; ESI-MS(+) m/z 942.6(M+2H).

ESI-HRMS(+) m/z:

Calculated: 941.9723 (M+2H).

Found: 941.9747 (M+2H).

Preparation of Example 3012

Example 3012 was prepared following the general synthetic sequencedescribed for the preparation of Example 3003, starting fromIntermediate Resin H, using the following general procedures:“Bromoacetic Acid Coupling” procedure; “N-Gly-Alkylation On-resin MethodA” procedure; “Symphony Method A: Secondary-amine coupling without Fmocdeprotection” procedure; “Symphony Method A: Standard couplingprocedure”; “Symphony Method A: Secondary-amine coupling procedure”;“Chloroacetic Anhydride capping” procedure; “Global Deprotection MethodA”; “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 45-85% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of product was 3.4 mg, and its estimated purityby LCMS analysis was 95% using “Analysis LCMS conditions D and E”.

Analysis LCMS condition D: Retention time=1.95 min; ESI-MS(+) m/z(M+2H), not detected.

Analysis LCMS condition E: Retention time=1.60 min; ESI-MS(+) m/z(M+2H), not detected.

ESI-HRMS(+) m/z:

Calculated: 942.4667 (M+2H).

Found: 942.4659 (M+2H).

Preparation of Example 3013

Example 3013 was prepared following the general synthetic sequencedescribed for the preparation of Example 3008, starting fromIntermediate Resin I, using the following general procedures:“Bromoacetic Acid Coupling” procedure; “N-Gly-Alkylation On-resin MethodC” procedure; “Symphony Method A: Secondary-amine coupling without Fmocdeprotection” procedure; Symphony Method A: Standard coupling”procedure; “Symphony Method A: Secondary-amine coupling” procedure;“Chloroacetic Anhydride capping” procedure; “Global Deprotection MethodA”; “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 45-85% B over 40minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of product was 5.8 mg, and its estimated purityby LCMS analysis was 97% using “Analysis LCMS conditions D and E”.

Analysis LCMS condition D: Retention time=1.80 min; ESI-MS(+) m/z 942.9(M+2H);

Analysis LCMS condition D: Retention time=1.56 min; ESI-MS(+) m/z 943.0(M+2H).

ESI-HRMS(+) m/z:

Calculated: 942.4667 (M+2H).

Found: 942.4656 (M+2H).

Preparation of Example 3014

Example 3014 was prepared following the general synthetic sequencedescribed for the preparation of Example 3008, starting fromIntermediate Resin I, using the following general procedures:“Bromoacetic Acid Coupling” procedure; “N-Gly-Alkylation On-resin MethodA” procedure; “Symphony Method A: Secondary-amine coupling without Fmocdeprotection” procedure; Symphony Method A: Standard coupling”procedure; “Symphony Method A: Secondary-amine coupling” procedure;“Chloroacetic Anhydride capping” procedure; “Global Deprotection MethodA”; “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 45-85% B over 40minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of product was 10.2 mg, and its estimated purityby LCMS analysis was 90% using “Analysis LCMS condition D”.

Analysis LCMS condition D: Retention time=1.75 min; ESI-MS(+) m/z 950.5(M+2H).

ESI-HRMS(+) m/z:

Calculated: 949.9722 (M+2H).

Found: 949.9704 (M+2H).

Preparation of Example 3015

Example 3015 was prepared following the general synthetic sequencedescribed for the preparation of Example 3008, starting fromIntermediate Resin I, using the following general procedures:“Bromoacetic Acid Coupling” procedure; “N-Gly-Alkylation On-resin MethodB” procedure; “Symphony Method A: Secondary-amine coupling without Fmocdeprotection” procedure; Symphony Method A: Standard coupling”procedure; “Symphony Method A: Secondary-amine coupling” procedure;“Chloroacetic Anhydride capping” procedure; “Global Deprotection MethodA”; “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 40-80% B over 40minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of product was 3.4 mg, and its estimated purityby LCMS analysis was 90% using “Analysis LCMS condition D”.

Analysis LCMS condition D: Retention time=1.72 min; ESI-MS(+) m/z 958.2(M+2H).

ESI-HRMS(+) m/z:

Calculated: 957.4538 (M+2H).

Found: 957.4521 (M+2H).

Preparation of Example 3016

Example 3016 was prepared following the general synthetic sequencedescribed for the preparation of Example 3008, starting fromIntermediate Resin I, using the following general procedures:“Bromoacetic Acid Coupling” procedure; “N-Gly-Alkylation On-resin MethodA” procedure; “Symphony Method A: Secondary-amine coupling without Fmocdeprotection” procedure; Symphony Method A: Standard coupling”procedure; “Symphony Method A: Secondary-amine coupling” procedure;“Chloroacetic Anhydride capping” procedure; “Global Deprotection MethodA”; “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 45-85% B over 35minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of product was 4.0 mg, and its estimated purityby LCMS analysis was 98% using “Analysis LCMS conditions D and E”.

Analysis LCMS condition D: Retention time=1.85 min; ESI-MS(+) m/z,(M+2H) not detected;

Analysis LCMS condition E: Retention time=1.56 min; ESI-MS(+) m/z,(M+2H) not detected.

ESI-HRMS(+) m/z:

Calculated: 950.4642 (M+2H).

Found: 950.4629 (M+2H).

Preparation of Example 3017

Example 3017 was prepared following the general synthetic sequencedescribed below, starting from Intermediate Resin E. The followingprocedures were performed sequentially:

“Nosylate formation”, “Alkylation method B”, “Nosylate removal”,“Symphony Method B: Custom amino acids-coupling procedure”, “SymphonyMethod B: Final capping procedure”, “Global Deprotection Method A”, and“Cyclization Method A”. The crude material was purified via preparativeLC/MS with the following conditions: Column: XBridge C18, 19×200 mm,5-μm particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammoniumacetate; Gradient: 45-85% B over 30 minutes, then a 5-minute hold at100% B; Flow: 20 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The material was furtherpurified via preparative LC/MS with the following conditions: Column:XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% B over30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 5.1 mg, and its estimatedpurity by LCMS analysis was 100%.

Analysis LCMS Condition H: retention time=1.71 min.; ESI-MS(+) m/z 956.2(M+2H);

Analysis LCMS Condition I: retention time=2.73 min.; ESI-MS(+) m/z 956.3(M+2H).

Preparation of Example 3019

Example 3019 was prepared following the general synthetic sequencedescribed for the preparation of Example 3017, starting fromIntermediate Resin E. The following procedures were performedsequentially: “Nosylate formation”, “Alkylation method A”, “Nosylateremoval”, “Symphony Method B: Custom amino acids-coupling procedure”,“Symphony Method B: Final capping procedure”, “Global DeprotectionMethod A”, and “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 45-85% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 10.7 mg, and its estimatedpurity by LCMS analysis was 97%.

Analysis LCMS Condition H: retention time=1.73 min.; ESI-MS(+) m/z 987.2(M+2H).

Analysis LCMS Condition I: retention time=2.81 min.; ESI-MS(+) m/z 987.2(M+2H).

Preparation of Example 3020

Example 3020 was prepared following the general synthetic sequencedescribed for the preparation of Example 3017, starting fromintermediate resin A. The following procedures were performedsequentially: “Nosylate formation”, “Alkylation method B”, “Nosylateremoval”, “Symphony Method B: Resin-swelling procedure”, “SymphonyMethod B: Standard-coupling procedure”, “Symphony Method B: Secondaryamine-coupling procedure”, “Symphony Method B: Custom aminoacids-coupling procedure”, “Symphony Method B: Final capping procedure”,“Global Deprotection Method A”, and “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 14.1 mg, and itsestimated purity by LCMS analysis was 97%.

Analysis LCMS Condition H: retention time=1.5 min.; ESI-MS(+) m/z 935.9(M+2H).

Analysis LCMS Condition J: retention time=1.26 min.; ESI-MS(+) m/z 935.0(M+2H).

Preparation of Example 3021

Example 3021 was prepared following the general synthetic sequencedescribed for the preparation of Example 3017, starting fromintermediate resin B. The following procedures were performedsequentially: “Nosylate formation”, “Alkylation method B”, “Nosylateremoval”, “Symphony Method B: Resin-swelling procedure”, “SymphonyMethod B: Standard-coupling procedure”, “Symphony Method B: Secondaryamine-coupling procedure”, “Symphony Method B: Custom aminoacids-coupling procedure”, “Symphony Method B: Final capping procedure”,“Global Deprotection Method A”, and “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile: water with 0.1% trifluoroacetic acid; Mobile PhaseB: 95:5 acetonitrile: water with 0.1% trifluoroacetic acid; Gradient:10-50% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20mL/min. Fractions containing the desired product were combined and driedvia centrifugal evaporation. The yield of the product was 6.0 mg, andits estimated purity by LCMS analysis was 100%.

Analysis LCMS Condition H: retention time=1.77 min.; ESI-MS(+) m/z 956.4(M+2H).

Analysis LCMS Condition J: retention time=1.47 min.; ESI-MS(+) m/z 956.3(M+2H).

Preparation of Example 3022

Example 3022 was prepared following the general synthetic sequencedescribed for the preparation of Example 3017, starting fromintermediate resin D. The following procedures were performedsequentially: “Nosylate formation”, “Alkylation method A”, “Nosylateremoval”, “Symphony Method B: Resin-swelling procedure”, “SymphonyMethod B: Standard-coupling procedure”, “Symphony Method B: Secondaryamine-coupling procedure”, “Symphony Method B: Custom aminoacids-coupling procedure”, “Symphony Method B: Final capping procedure”,“Global Deprotection Method A”, and “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 35-75% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 10.7 mg, and its estimatedpurity by LCMS analysis was 100%.

Analysis LCMS Condition H: retention time=1.26 min.; ESI-MS(+) m/z 942.8(M+2H).

Analysis LCMS Condition J: retention time=1.0 min.; ESI-MS(+) m/z 942.5(M+2H).

Preparation of Example 3023

Example 3023 was prepared following the general synthetic sequencedescribed for the preparation of Example 3017, starting fromintermediate resin C. The following procedures were performedsequentially: “Nosylate formation”, “Alkylation method B”, “Nosylateremoval”, “Symphony Method B: Resin-swelling procedure”, “SymphonyMethod B: Standard-coupling procedure”, “Symphony Method B: Secondaryamine-coupling procedure”, “Symphony Method B: Custom aminoacids-coupling procedure”, “Symphony Method B: Final capping procedure”,“Global Deprotection Method A”, and “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 6.9 mg, and itsestimated purity by LCMS analysis was 98%.

Analysis LCMS Condition H: retention time=1.67 min.; ESI-MS(+) m/z 955.9(M+2H).

Analysis LCMS Condition I: retention time=2.73 min.; ESI-MS(+) m/z 956.2(M+2H).

Preparation of Example 3024

Example 3024 was prepared following the general synthetic sequencedescribed for the preparation of Example 3017, starting fromintermediate resin D. The following procedures were performedsequentially: “Nosylate formation”, “Alkylation method B”, “Nosylateremoval”, “Symphony Method B: Resin-swelling procedure”, “SymphonyMethod B: Standard-coupling procedure”, “Symphony Method B: Secondaryamine-coupling procedure”, “Symphony Method B: Custom aminoacids-coupling procedure”, “Symphony Method B: Final capping procedure”,“Global Deprotection Method A”, and “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 9.2 mg, and itsestimated purity by LCMS analysis was 100%.

Analysis LCMS Condition H: retention time=1.59 min.; ESI-MS(+) m/z 935.0(M+2H).

Analysis LCMS Condition E: retention time=1.38 min.; ESI-MS(+) m/z 935.3(M+2H).

Preparation of Example 3025

Example 3025 was prepared following the general synthetic sequencedescribed for the preparation of Example 3017, starting fromintermediate resin D. The following procedures were performedsequentially: “Nosylate formation”, “Alkylation method A”, “Nosylateremoval”, “Symphony Method B: Resin-swelling procedure”, “SymphonyMethod B: Standard-coupling procedure”, “Symphony Method B: Secondaryamine-coupling procedure”, “Symphony Method B: Custom aminoacids-coupling procedure”, “Symphony Method B: Final capping procedure”,“Global Deprotection Method A”, and “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 8.0 mg, and itsestimated purity by LCMS analysis was 95%.

Analysis LCMS Condition H: retention time=1.5 min.; ESI-MS(+) m/z 950.0(M+2H).

Analysis LCMS Condition J: retention time=1.37 min.; ESI-MS(+) m/z 949.6(M+2H).

Preparation of Example 3026

Example 3026 was prepared following the general synthetic sequencedescribed for the preparation of Example 3017, starting fromintermediate resin E. The following procedures were performedsequentially: “Nosylate formation”, “Alkylation method A”, “Nosylateremoval”, “Symphony Method B: Custom amino acids-coupling procedure”,“Symphony Method B: Final capping procedure”, “Global DeprotectionMethod A”, and “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The material was further purified viapreparative LC/MS with the following conditions: Column: Waters CSH C18,19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with10-mM ammonium acetate; Gradient: 5-45% B over 30 minutes, then a5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 10.8 mg, and its estimated purity by LCMSanalysis was 78%.

Analysis LCMS Condition H: retention time=1.74 min.; ESI-MS(+) m/z 971.1(M+2H).

Analysis LCMS Condition J: retention time=1.37 min.; ESI-MS(+) m/z 971.1(M+2H).

Preparation of Example 3028

Example 3028 was prepared following the general synthetic sequencedescribed for the preparation of Example 3017, starting fromintermediate resin D. The following procedures were performedsequentially: “Nosylate formation”, “Alkylation method B”, “Nosylateremoval”, “Symphony Method B: Resin-swelling procedure”, “SymphonyMethod B: Standard-coupling procedure”, “Symphony Method B: Secondaryamine-coupling procedure”, “Symphony Method B: Custom aminoacids-coupling procedure”, “Symphony Method B: Final capping procedure”,“Global Deprotection Method A”, and “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 5-45% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 4.1 mg, and itsestimated purity by LCMS analysis was 82%.

Analysis LCMS Condition H: retention time=1.31 min.; ESI-MS(+) m/z 950.0(M+2H).

Analysis LCMS Condition J: retention time=1.16 min.; ESI-MS(+) m/z 950.0(M+2H).

Preparation of Example 3029

Example 3029 was prepared following the general synthetic sequencedescribed for the preparation of Example 3017, starting fromintermediate resin D. The following procedures were performedsequentially: “Nosylate formation”, “Alkylation method A”, “Nosylateremoval”, “Symphony Method B: Resin-swelling procedure”, “SymphonyMethod B: Standard-coupling procedure”, “Symphony Method B: Secondaryamine-coupling procedure”, “Symphony Method B: Custom aminoacids-coupling procedure”, “Symphony Method B: Final capping procedure”,“Global Deprotection Method A”, and “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 50-90% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 2.7 mg, and its estimatedpurity by LCMS analysis was 78%.

Analysis LCMS Condition H: retention time=1.97 min.; ESI-MS(+) m/z 967.7(M+2H).

Analysis LCMS Condition J: retention time=1.53 min.; ESI-MS(−) m/z 965.5(M+2H).

Preparation of Example 3030

Example 3030 was prepared following the general synthetic sequencedescribed for the preparation of Example 3017, starting fromintermediate resin C. The following procedures were performedsequentially: “Nosylate formation”, “Alkylation method A”, “Nosylateremoval”, “Symphony Method B: Resin-swelling procedure”, “SymphonyMethod B: Standard-coupling procedure”, “Symphony Method B: Secondaryamine-coupling procedure”, “Symphony Method B: Custom aminoacids-coupling procedure”, “Symphony Method B: Final capping procedure”,“Global Deprotection Method A”, and “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% Bover 40 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 3.0 mg, and itsestimated purity by LCMS analysis was 86%.

Analysis LCMS Condition H: retention time=1.59 min.; ESI-MS(+) m/z 964.3(M+2H).

Analysis LCMS Condition J: retention time=1.27 min.; ESI-MS(+) m/z 964.3(M+2H).

Preparation of Example 3032

Example 3032 was prepared following the general synthetic sequencedescribed for the preparation of Example 3017, starting fromintermediate resin E. The following procedures were performedsequentially: “Nosylate formation”, “Alkylation method A”, “Nosylateremoval”, “Symphony Method B: Custom amino acids-coupling procedure”,“Symphony Method B: Final capping procedure”, “Global DeprotectionMethod A”, and “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The material was further purified viapreparative LC/MS with the following conditions: Column: Waters CSH C18,19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with10-mM ammonium acetate; Gradient: 0-40% B over 30 minutes, then a5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 4.6 mg, and its estimated purity by LCMSanalysis was 98%.

Analysis LCMS Condition H: retention time=1.69 min.; ESI-MS(+) m/z 963.4(M+2H).

Analysis LCMS Condition J: retention time=1.14 min.; ESI-MS(+) m/z 964.2(M+2H).

Preparation of Example 3033

Example 3033 was prepared following the general synthetic sequencedescribed for the preparation of Example 3017, starting fromIntermediate resin D. The following procedures were performedsequentially: “Nosylate formation”, “Alkylation method A”, “Nosylateremoval”, “Symphony Method B: Resin-swelling procedure”, “SymphonyMethod B: Standard-coupling procedure”, “Symphony Method B: Secondaryamine-coupling procedure”, “Symphony Method B: Custom aminoacids-coupling procedure”, “Symphony Method B: Final capping procedure”,“Global Deprotection Method A”, and “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 5-45% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The material was further purified viapreparative LC/MS with the following conditions: Column: XBridge C18,19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol:water with10-mM ammonium acetate; Mobile Phase B: 95:5 methanol: water with 10-mMammonium acetate; Gradient: 25-70% B over 30 minutes, then a 5-minutehold at 100% B; Flow: 20 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. The yieldof the product was 0.6 mg, and its estimated purity by LCMS analysis was85%.

Analysis LCMS Condition H: retention time=1.42 min.; ESI-MS(+) m/z 943.0(M+2H).

Analysis LCMS Condition J: retention time=1.13 min.; ESI-MS(+) m/z1883.9 (M+H).

Preparation of Example 3037

Example 3037 was prepared following the general synthetic sequencedescribed for the preparation of Example 3017, starting withIntermediate resin F. The following procedures were performedsequentially: “N-Gly-Alkylation On-resin Method D”, “Symphony Method B:Resin-swelling procedure”, “Symphony Method B: Standard-couplingprocedure”, “Symphony Method B: Secondary amine-coupling procedure”,“Symphony Method B: Custom amino acids-coupling procedure”, “SymphonyMethod B: Final capping procedure”, “Global Deprotection Method A”, and“Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 0-40% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 32.0 mg, and itsestimated purity by LCMS analysis was 94%.

Analysis LCMS Condition H: retention time=1.25 min.; ESI-MS(+) m/z 943.2(M+2H);

Analysis LCMS Condition J: retention time=1.14 min.; ESI-MS(+) m/z 943.2(M+2H).

Preparation of Example 3038

Example 3038 was prepared following the general synthetic sequencedescribed for the preparation of Example 3017, composed of the followinggeneral procedures, starting with intermediate resin F:“N-Gly-Alkylation On-resin Method D”, “Symphony Method B: Resin-swellingprocedure”, “Symphony Method B: Standard-coupling procedure”, “SymphonyMethod B: Secondary amine-coupling procedure”, “Symphony Method B:Custom amino acids-coupling procedure”, “Symphony Method B: Finalcapping procedure”, “Global Deprotection Method A”, and “CyclizationMethod A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 11.9 mg, and itsestimated purity by LCMS analysis was 95%.

Analysis LCMS Condition H: retention time=1.64 min.; ESI-MS(+) m/z 962.1(M+2H);

Analysis LCMS Condition J: retention time=1.33 min.; ESI-MS(+) m/z 962.0(M+2H).

Preparation of Example 3041

Example 3041 was prepared following the general synthetic sequencedescribed for the preparation of Example 3017, composed of the followinggeneral procedures, starting from intermediate resin D: “Nosylateformation”, “Alkylation method A”, “Nosylate removal”, “Symphony MethodB: Resin-swelling procedure”, “Symphony Method B: Standard-couplingprocedure”, “Symphony Method B: Secondary amine-coupling procedure”,“Symphony Method B: Custom amino acids-coupling procedure”, “SymphonyMethod B: Final capping procedure”, “Global Deprotection Method A”, and“Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 3.8 mg, and itsestimated purity by LCMS analysis was 81%.

Analysis LCMS Condition H: retention time=1.68 min.; ESI-MS(+) m/z 970.0(M+2H);

Analysis LCMS Condition J: retention time=1.51 min.; ESI-MS(+) m/z 969.3(M+2H).

Preparation of Example 3044

Example 3044 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures, starting with intermediate resin G:“N-Gly-Alkylation On-resin Method D”, “Symphony Method B: Custom aminoacids-coupling procedure”, “Symphony Method B: Final capping procedure”,“Global Deprotection Method A”, and “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: waters CSH c-18, 19×200 mm, 5-μm particles; MobilePhase A: 5:95 acetonitrile: water with 0.1% trifluoroacetic acid; MobilePhase B: 95:5 acetonitrile: water with 0.1% trifluoroacetic acid;Gradient: 10-50% B over 30 minutes, then a 5-minute hold at 100% B;Flow: 20 mL/min. Fractions containing the desired product were combinedand dried via centrifugal evaporation. The material was further purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: waterwith 10-mM ammonium acetate; Gradient: 10-50% B over 30 minutes, then a5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 19.4 mg, and its estimated purity by LCMSanalysis was 96%.

Analysis LCMS Condition H: retention time=1.55 min.; ESI-MS(+) m/z 964.1(M+2H);

Analysis LCMS Condition J: retention time=1.32 min.; ESI-MS(+) m/z 963.8(M+2H).

Preparation of Example 3045

Example 3045 was prepared following the general synthetic sequencedescribed for the preparation of Example 3017, composed of the followinggeneral procedures, starting with intermediate resin G:“N-Gly-Alkylation On-resin Method D”, “Symphony Method B: Custom aminoacids-coupling procedure”, “Symphony Method B: Final capping procedure”,“Global Deprotection Method A”, and “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 15-55% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The material was further purified viapreparative LC/MS with the following conditions: Column: XBridge C18,19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with10-mM ammonium acetate; Gradient: 5-45% B over 30 minutes, then a5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 1.5 mg, and its estimated purity by LCMSanalysis was 94%.

Analysis LCMS Condition H: retention time=1.60 min.; ESI-MS(+) m/z 957.1(M+2H);

Analysis LCMS Condition J: retention time=1.30 min.; ESI-MS(+) m/z 957.5(M+2H).

Preparation of Example 3046

Example 3046 was prepared following the general synthetic sequencedescribed for the preparation of Example 3017, composed of the followinggeneral procedures, starting with intermediate resin G:“N-Gly-Alkylation On-resin Method D”, “Symphony Method B: Custom aminoacids-coupling procedure”, “Symphony Method B: Final capping procedure”,“Global Deprotection Method A”, and “Cyclization Method A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The material was further purified viapreparative LC/MS with the following conditions: Column: XBridge C18,19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with10-mM ammonium acetate; Gradient: 10-50% B over 30 minutes, then a5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 7.2 mg, and its estimated purity by LCMSanalysis was 100%.

Analysis LCMS Condition H: retention time=1.71 min.; ESI-MS(+) m/z 982.9(M+2H).

Analysis LCMS Condition J: retention time=1.40 min.; ESI-MS(+) m/z 982.9(M+2H).

Preparation of Example 3047

Example 3047 was prepared following the general synthetic sequencedescribed for the preparation of Example 3017, composed of the followinggeneral procedures, starting with intermediate resin F:“N-Gly-Alkylation On-resin Method D”, “Symphony Method B: Resin-swellingprocedure”, “Symphony Method B: Standard-coupling procedure”, “SymphonyMethod B: Secondary amine-coupling procedure”, “Symphony Method B:Custom amino acids-coupling procedure”, “Symphony Method B: Finalcapping procedure”, “Global Deprotection Method A”, and “CyclizationMethod A”.

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 40-80% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The material was further purified via preparative LC/MSwith the following conditions: Column: Waters CSH C18, 19×200 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammoniumacetate; Gradient: 5-45% B over 30 minutes, then a 5-minute hold at 100%B; Flow: 20 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 1.0 mg, and its estimated purity by LCMS analysis was 97%.

Analysis LCMS Condition H: retention time=1.44 min.; ESI-MS(+) m/z 936.1(M+2H);

Analysis LCMS Condition J: retention time=1.21 min.; ESI-MS(+) m/z 936.4(M+2H).

Experimental Procedures for Compounds 5001, 5002, 5003 Analytical Data:

Mass Spectrometry: “ESI-MS(+)” signifies electrospray ionization massspectrometry performed in positive ion mode; “ESI-MS(−)” signifieselectrospray ionization mass spectrometry performed in negative ionmode; “ESI-HRMS(+)” signifies high-resolution electrospray ionizationmass spectrometry performed in positive ion mode; “ESI-HRMS(−)”signifies high-resolution electrospray ionization mass spectrometryperformed in negative ion mode. The detected masses are reportedfollowing the “m/z” unit designation. Compounds with exact massesgreater than 1000 were often detected as double-charged ortriple-charged ions.

Analysis Condition A:

Column: Waters BEH C18, 2.0×50 mm, 1.7-μm particles; Mobile Phase A:5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 50°C.; Gradient: 0% B, 0-100% B over 3 minutes, then a 0.5-minute hold at100% B; Flow: 1 mL/min; Detection: UV at 220 nm.

Analysis Condition B:

Column: Waters BEH C18, 2.0×50 mm, 1.7-μm particles; Mobile Phase A:5:95 methanol:water with 10 mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10 mM ammonium acetate; Temperature: 50° C.;Gradient: 0% B, 0-100% B over 3 minutes, then a 0.5-minute hold at 100%B; Flow: 0.5 mL/min; Detection: UV at 220 nm.

Analysis Condition C:

Column: Waters Aquity BEH C18 2.1×50 mm 1.7 μm particles; Mobile PhaseA: water with 0.05% TFA; Mobile Phase B: acetonitrile with 0.05% TFA;Temperature: 40° C.; Gradient: 0% B, 0-100% B over 1.5 minutes, then a0.5-minute hold at 100% B; Flow: 0.8 mL/min; Detection: UV at 220 nm.

General Procedures: Prelude Method A:

All manipulations were performed under automation on a Prelude peptidesynthesizer (Protein Technologies). All procedures unless noted wereperformed in a 10 mL polypropylene tube fitted with a bottom frit; wherethe scale of the reaction exceeded 0.100 mmol, a 40 mL polypropylenetube fitted with a bottom frit was used. The tube connects to a thePrelude peptide synthesizer through both the bottom and the top of thetube. DMF and DCM can be added through the top of the tube, which washesdown the sides of the tube equally. The remaining reagents are addedthrough the bottom of the tube and pass up through the fit to contactthe resin. All solutions are removed through the bottom of the tube.“Periodic agitation” describes a brief pulse of N2 gas through thebottom frit; the pulse lasts approximately 5 seconds and occurs every 30seconds. Chloroacetyl chloride solutions in DMF were used within 24 h ofpreparation. Amino acid solutions were generally not used beyond threeweeks from preparation. HATU solutions were used within 5 days ofpreparation. DMF=dimethylformamide;HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate; DIPEA=diisopropylethylamine;Rink=(2,4-dimethoxyphenyl)(4-alkoxyphenyl)methanamine, where “4-alkoxy”describes the position and type of connectivity to the polystyreneresin. The resin used is Merrifield polymer (polystyrene) with a Rinklinker (Fmoc-protected at nitrogen); 100-200 mesh, 1% DVB, 0.56 mmol/gloading. Common amino acids used are listed below with side-chainprotecting groups indicated inside parenthesis.

Fmoc-Ala-OH; Fmoc-Arg(Pbf)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Asp(OtBu)-OH;Fmoc-Bzt-OH; Fmoc-Cys(Trt)-OH; Fmoc-Dab(Boc)-OH; Fmoc-Dap(Boc)-OH;Fmoc-Gln(Trt)-OH; Fmoc-Gly-OH; Fmoc-His(Trt)-OH; Fmoc-Hyp(tBu)-OH;Fmoc-Ile-OH; Fmoc-Leu-OH; Fmoc-Lys(Boc)-OH; Fmoc-Nle-OH; Fmoc-Met-OH;Fmoc-[N-Me]Ala-OH; Fmoc[N-Me]Nle-OH; Fmoc-Phe-OH; Fmoc-Pro-OH;Fmoc-Sar-OH; Fmoc-Ser(tBu)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Trp(Boc)-OH;Fmoc-Tyr(tBu)-OH; Fmoc-Val-OH.

The procedures of “Prelude Method A” describe an experiment performed ona 0.100 mmol scale, where the scale is determined by the amount of Rinklinker bound to the resin. This scale corresponds to approximately 178mg of the Rink-Merrifield resin described above. All procedures can bescaled beyond 0.100 mmol scale by adjusting the described volumes by themultiple of the scale. Prior to amino acid coupling, all peptidesynthesis sequences began with a resin-swelling procedure, describedbelow as “Resin-swelling procedure”. Coupling of amino acids to aprimary amine N-terminus used the “Single-coupling procedure” describedbelow. Coupling of amino acids to a secondary amine N-terminus used the“Double-coupling procedure” described below. Coupling ofchloroacetylchloride to the N-terminus of the peptide is described bythe “Chloroacetyl chloride coupling procedure” detailed below.

Resin-Swelling Procedure:

To a 10 mL polypropylene solid-phase reaction vessel was addedMerrifield: Rink resin (178 mg, 0.100 mmol). The resin was washed(swelled) three times as follows: to the reaction vessel was added DMF(2.0 mL), upon which the mixture was periodically agitated for 10minutes before the solvent was drained through the frit.

Single-Coupling Procedure:

To the reaction vessel containing resin from the previous step was addedpiperidine:DMF (20:80 v/v, 2.0 mL). The mixture was periodicallyagitated for 3 minutes and then the solution was drained through thefrit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.0mL). The mixture was periodically agitated for 3 minutes and then thesolution was drained through the frit. The resin was washed successivelysix times as follows: for each wash, DMF (2.0 mL) was added through thetop of the vessel and the resulting mixture was periodically agitatedfor 30 seconds before the solution was drained through the frit. To thereaction vessel was added the amino acid (0.2M in DMF, 1.0 mL, 2 eq),then HATU (0.2M in DMF, 1.0 mL, 2 eq), and finally DIPEA (0.8M in DMF,0.5 mL, 4 eq). The mixture was periodically agitated for 15 minutes,then the reaction solution was drained through the frit. The resin waswashed successively four times as follows: for each wash, DMF (2.0 mL)was added through the top of the vessel and the resulting mixture wasperiodically agitated for 30 seconds before the solution was drainedthrough the frit. To the reaction vessel was added acetic anhydride (2.0mL). The mixture was periodically agitated for 10 minutes, then thesolution was drained through the frit. The resin was washed successivelyfour times as follows: for each wash, DMF (2.0 mL) was added through thetop of the vessel and the resulting mixture was periodically agitatedfor 90 seconds before the solution was drained through the frit. Theresulting resin was used directly in the next step.

Double-Coupling Procedure:

To the reaction vessel containing resin from the previous step was addedpiperidine:DMF (20:80 v/v, 2.0 mL). The mixture was periodicallyagitated for 3 minutes and then the solution was drained through thefrit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.0mL). The mixture was periodically agitated for 3 minutes and then thesolution was drained through the frit. The resin was washed successivelysix times as follows: for each wash, DMF (2.0 mL) was added through thetop of the vessel and the resulting mixture was periodically agitatedfor 30 seconds before the solution was drained through the frit. To thereaction vessel was added the amino acid (0.2M in DMF, 1.0 mL, 2 eq),then HATU (0.2M in DMF, 1.0 mL, 2 eq), and finally DIPEA (0.8M in DMF,0.5 mL, 4 eq). The mixture was periodically agitated for 15 minutes,then the reaction solution was drained through the frit. The resin wastwice washed as follows: for each wash, DMF (2.0 mL) was added throughthe top of the vessel and the resulting mixture was periodicallyagitated for 30 seconds before the solution was drained through thefrit. To the reaction vessel was added the amino acid (0.2M in DMF, 1.0mL, 2 eq), then HATU (0.2M in DMF, 1.0 mL, 2 eq), and finally DIPEA(0.8M in DMF, 0.5 mL, 4 eq). The mixture was periodically agitated for15 minutes, then the reaction solution was drained through the frit. Theresin was twice washed as follows: for each wash, DMF (2.0 mL) was addedthrough the top of the vessel and the resulting mixture was periodicallyagitated for 30 seconds before the solution was drained through thefrit. To the reaction vessel was added acetic anhydride (2.0 mL). Themixture was periodically agitated for 10 minutes, then the solution wasdrained through the frit. The resin was washed successively four timesas follows: for each wash, DMF (2.0 mL) was added through the top of thevessel and the resulting mixture was periodically agitated for 90seconds before the solution was drained through the frit. The resultingresin was used directly in the next step.

Chloroacetyl Chloride Coupling Procedure:

To the reaction vessel containing the resin from the previous step wasadded piperidine:DMF (20:80 v/v, 2.0 mL). The mixture was periodicallyagitated for 3 minutes and then the solution was drained through thefrit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.0mL). The mixture was periodically agitated for 3 minutes and then thesolution was drained through the frit. The resin was washed successivelysix times as follows: for each wash, DMF (2.0 mL) was added through thetop of the vessel and the resulting mixture was periodically agitatedfor 30 seconds before the solution was drained through the frit. To thereaction vessel was added DIPEA (0.8M in DMF, 3.0 mL, 24 eq), thenchloroacetyl chloride (0.8M in DMF, 1.65 mL, 13.2 eq). The mixture wasperiodically agitated for 30 minutes, then the solution was drainedthrough the frit. The resin was washed successively three times asfollows: for each wash, DMF (2.0 mL) was added to top of the vessel andthe resulting mixture was periodically agitated for 90 seconds beforethe solution was drained through the frit. The resin was washedsuccessively four times as follows: for each wash, CH₂C₁₂ (2.0 mL) wasadded to top of the vessel and the resulting mixture was periodicallyagitated for 90 seconds before the solution was drained through thefrit. The resulting resin was placed under a N₂ stream for 15 minutes.

Symphony Method A:

This collection of procedures is identical that of “Prelude Method A”except as noted. For all procedures a Symphony X peptide synthesizer(Protein Technologies) was used instead of a Prelude peptide synthesizerand all reagents were added through the top of the reaction vessel.

Resin-Swelling Procedure:

This procedure is identical to “Prelude Method A: Resin-swellingprocedure”.

Single-Coupling Procedure:

This procedure is identical to “Prelude Method A: Single-couplingprocedure” except that the concentration of DIPEA solution was 0.4M and1.0 mL of this solution was delivered to the reaction.

Double-Coupling Procedure:

This procedure is identical to “Prelude Method A: Double-couplingprocedure” except that the concentration of DIPEA solution was 0.4M and1.0 mL of this solution was delivered to the reaction.

Chloroacetyl Chloride Coupling Procedure:

This procedure is identical to “Prelude Method A: Chloroacetyl chloridecoupling procedure”.

Global Deprotection Method A:

All manipulations were performed manually unless noted. The procedure of“Global Deprotection Method A” describes an experiment performed on a0.100 mmol scale, where the scale is determined by the amount of Rinklinker bound to the resin. The procedure can be scaled beyond 0.100 mmolscale by adjusting the described volumes by the multiple of the scale. A“deprotection solution” was prepared by combining in a 40 mL glass vialtrifluoroacetic acid (22 mL), phenol (1.325 g), water (1.25 mL) andtriisopropylsilane (0.5 mL). The resin was removed from the reactionvessel and transferred to a 4 mL glass vial. To the vial was added the“deprotection solution” (2.0 mL). The mixture was vigorously mixed in ashaker (1000 RPM for 1 minute, then 500 RPM for 1-2 h). The mixture wasfiltered through a 0.2 micron syringe filter and the solids wereextracted with the “deprotection solution” (1.0 mL) or TFA (1.0 mL). Toa 24 mL test tube charged with the combined filtrates was added Et₂O (15mL). The mixture was vigorously mixed upon which a significant amount ofa white solid precipitated. The mixture was centrifuged for 5 minutes,then the solution was decanted away from the solids and discarded. Thesolids were suspended in Et₂O (20 mL); then the mixture was centrifugedfor 5 minutes; and the solution was decanted away from the solids anddiscarded. For a final time, the solids were suspended in Et₂O (20 mL);the mixture was centrifuged for 5 minutes; and the solution was decantedaway from the solids and discarded to afford the crude peptide as awhite to off-white solid.

Cyclization Method A:

All manipulations were performed manually unless noted. The procedure of“Cyclization Method B” describes an experiment performed on a 0.100 mmolscale, where the scale is determined by the amount of Rink linker boundto the resin that was used to generate the peptide. This scale is notbased on a direct determination of the quantity of peptide used in theprocedure. The procedure can be scaled beyond 0.100 mmol scale byadjusting the described volumes by the multiple of the scale. The crudepeptide solids were dissolved in MeCN:aq. 0.1M NH₄OAc (1:1) to a totalvolume of 18-22 mL, and the solution was carefully then adjusted topH=8.5-9.0 using aq NaOH (1.0M). The solution was then allowed to standwithout stirring for 6 days. The reaction solution was concentrated andthe residue was then dissolved in DMSO:MeOH. This solution was subjectedto reverse-phase HPLC purification to afford the desired cyclic peptide.

General Synthetic Sequence A:

“General Synthetic Sequence A” describes a general sequence ofprocedures that were used to afford the cyclic peptides describedherein. For the purposes of this general procedure, the procedures of“Symphony Method A” are interchangeable with those of “Prelude MethodA”. To a 10 mL polypropylene solid-phase reaction vessel was addedRink-Merrifield resin (178 mg, 0.100 mmol), and the reaction vessel wasplaced on the Prelude peptide synthesizer. “Prelude Method A:Resin-swelling procedure” was followed. Then a series of amino acidscouplings was sequentially performed on the Prelude following “PreludeMethod A: Single-coupling procedure” if the N-terminus of theresin-bound peptide was a primary amine or “Prelude Method A:Double-coupling procedure” if the N-terminus of the resin-bound peptidewas a secondary amine. “Prelude Method A: Chloroacetyl chloride couplingprocedure” was followed; then “Global Deprotection Method A” wasfollowed; then “Cyclization Method A” was followed.

Preparation of Example 5001

Example 5001 was prepared following “General Synthetic Sequence A. Inthe synthesis2-((((9H-fluoren-9-yl)methoxy)carbonyl)(2-(tert-butoxy)-2-oxoethyl)amino)aceticacid was used as indicated by the sequence. The crude material waspurified via preparative LC/MS with the following conditions: Column:XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 50-90% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 7.8 mg, and its estimatedpurity by LCMS analysis was 100%.

Analysis condition A: Retention time=1.48 min; ESI-MS(+) m/z 956.8.1(M−2H)

Analysis condition B: Retention time=2.82 min; ESI-MS(+) m/z 958.2(M+2H).

Preparation of Example 5002

Example 5002 was prepared following “General Synthetic Sequence A”. Inthe synthesis2-((((9H-fluoren-9-yl)methoxy)carbonyl)(2-(tert-butoxy)-2-oxoethyl)amino)aceticacid was used as indicated by the sequence. The crude material waspurified via preparative LC/MS with the following conditions: Column:XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 55-95% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 2.2 mg, and its estimatedpurity by LCMS analysis was 81%.

Analysis condition A: Retention time=1.51 min; ESI-MS(+) m/z 964.1(M−2H)

Analysis condition B: Retention time=2.88 min; ESI-MS(+) m/z 966.5(M+2H).

Preparation of Example 5003

Example 5003 was prepared following “General Synthetic Sequence A”. Inthe synthesis2-((((9H-fluoren-9-yl)methoxy)carbonyl)(2-(tert-butoxy)-2-oxoethyl)amino)aceticacid was used as indicated by the sequence. The crude material waspurified via preparative LC/MS with the following conditions: Column:XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 60-100% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The material was further purified via preparative LC/MSwith the following conditions: Column: XBridge Phenyl, 19×200 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammoniumacetate; Gradient: 5-40% B over 30 minutes, then a 5-minute hold at 100%B; Flow: 20 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 4.0 mg, and its estimated purity by LCMS analysis was 100%.

Analysis condition A: Retention time=1.777 min; ESI-MS(+) m/z 972.40(M+2H)

Analysis condition B: Retention time=2.393 min; ESI-MS(+) m/z 968.25(M−2H)

ESI-HRMS(+) m/z: Calculated: 971.9344 (M+2H). Found: 971.9311 (M+2H).

Methods for Testing the Ability of Macrocyclic Peptides to Compete forthe Binding of Pd-1 to Pd-L1 Using Homogenous Time-Resolved Fluorescence(HTRF) Binding Assays

The ability of the macrocyclic peptides of the present disclosure tobind to PD-L1 was investigated using a PD-1/PD-L1 HomogenousTime-Resolved Fluorescence (HTRF) binding assay.

Methods

Homogenous Time-Resolved Fluorescence (HTRF) Assays of Binding ofSoluble PD-1 to Soluble PD-L1. Soluble PD-1 and soluble PD-L1 refers toproteins with carboxyl-end truncations that remove thetransmembrane-spanning regions and are fused to heterologous sequences,specifically the Fc portion of the human immunoglobuling G sequence (Ig)or the hexahistidine epitope tag (His). All binding studies wereperformed in an HTRF assay buffer consisting of dPBS supplemented with0.1% (w/v) bovine serum albumin and 0.05% (v/v) Tween-20. For thePD-1-Ig/PD-L1-His binding assay, inhibitors were pre-incubated withPD-L1-His (10 nM final) for 15 m in 4 μl of assay buffer, followed byaddition of PD-1-Ig (20 nM final) in 1 μl of assay buffer and furtherincubation for 15 m. PD-L1 fusion proteins from either human,cynomologous macaques, mouse, or other species were used. HTRF detectionwas achieved using europium crypate-labeled anti-Ig monoclonal antibody(1 nM final) and allophycocyanin (APC) labeled anti-His monoclonalantibody (20 nM final). Antibodies were diluted in HTRF detection bufferand 5 μl was dispensed on top of binding reaction. The reaction wasallowed to equilibrate for 30 minutes and signal (665 nm/620 nm ratio)was obtained using an EnVision fluorometer. Additional binding assayswere established between PD-1-Ig/PD-L2-His (20 and 5 nM, respectively),CD80-His/PD-L1-Ig (100 and 10 nM, respectively) and CD80-His/CTLA4-Ig(10 and 5 nM, respectively). Binding/competition studies betweenbiotinylated Compound No. 71 and human PD-L1-His were performed asfollows. Macrocyclic peptide inhibitors were pre-incubated withPD-L1-His (10 nM final) for 60 minutes in 4 μl of assay buffer followedby addition of biotinylated Compound No. 71 (0.5 nM final) in 1 μl ofassay buffer. Binding was allowed to equilibrate for 30 minutes followedby addition of europium crypated labeled Streptavidin (2.5 pM final) andAPC-labeled anti-His (20 nM final) in 5 μl of HTRF buffer. The reactionwas allowed to equilibrate for 30 m and signal (665 nm/620 nm ratio) wasobtained using an EnVision fluorometer.

Recombinant Proteins. Carboxyl-truncated human PD-1 (amino acids 25-167)with a C-terminal human Ig epitope tag [hPD-1 (25-167)-3S-IG] and humanPD-L1 (amino acids 18-239) with a C-terminal His epitope tag[hPD-L1(19-239)-tobacco vein mottling virus protease cleavage site(TVMV)-His] were expressed in HEK293T cells and purified sequentially byrecombinant Protein A affinity chromatography and size exclusionchromatography. Human PD-L2-His (Sino Biologicals), CD80-His (SinoBiologicals), CTLA4-Ig (RnD Systems) were all obtained throughcommercial sources.

Sequence of Recombinant Human PD-1-Ig

hPD1(25-167)-3S-IG

(SEQ ID NO: 1) 1 LDSPDRPWNP PTFSPALLVV TEGDNATFTC SFSNTSESFV LNWYRMSPSN51 QTDKLAAFPE DRSQPGQDCR FRVTQLPNGR DFHMSVVEAR RNDSGTYLCG 101AISLAPKAQI KESLRAELRV TERRAEVPTA HPSPSPRPAG QFQGSPGGGG 151GREPKSSDKT HTSPPSPAPE LLGGSSVFLF PPKPKDTLMI SPTPEVTCVV 201VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE SQYNSTYRVV SVLTVLHQDW 251LNGKEYKCKV SNKALPAPIE KTISKAKGQP PEPQVYTLPP SRDELTKNQV 301SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD 351KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK

Sequence of Recombinant Human PD-L1-TVMV-His (PD-L1-His)

hPDL1(19-239)-TVMV-His

(SEQ ID NO: 2) 1 FTVTVPKDLY VVEYGSNMTI ECKFPVEKQL DLAALIVYWE MEDKNIIQFV51 HGEEDLKVQH SSYRQRARLL KDQLSLGNAA LQITDVKLQD AGVYRCMISY 101GGADYKRITV KVNAPYNKIN QRILVVDPVT SEHELTCQAE GYPKAEVIWT 151SSDHQVLSGK TTTTNSKREE KLFNVTSTLR INTTTNEIFY CTFRRLDPEE 201NHTAELVIPE LPLAHPPNER TGSSETVRFQ GHHHHHH

The results are shown in Table 1. As shown, the macrocyclic peptides ofthe present disclosure demonstrated potent inhibition of PD-1-Ig bindingactivity to PD-L1-TVMV-His (PD-L1-His). Ranges are as follows: A=0.10-10μM; B=0.01-0.099 μM; C=0.005-0.0099 μM.

TABLE 1 Example Number HTRF IC50 (μM) 3003 B 3004 B 3005 0.03 3006 B3007 B 3008 0.20 3009 B 3010 B 3011 6.12E−03 3012 B 3013 B 3014 B 3015 B3016 B 3017 C 3019 B 3020 C 3021 C 3022 B 3023 B 3024 B 3025 B 3026 B3028 A 3029 A 3030 B 3032 B 3033 A 3037 A 3038 2.36 3041 B 3044 B 3045 B3046 B 3047 — 3048 A 3049 A 3050 A 5001 C 5002 C 5003 B

It will be evident to one skilled in the art that the present disclosureis not limited to the foregoing illustrative examples, and that it canbe embodied in other specific forms without departing from the essentialattributes thereof. It is therefore desired that the examples beconsidered in all respects as illustrative and not restrictive,reference being made to the appended claims, rather than to theforegoing examples, and all changes which come within the meaning andrange of equivalency of the claims are therefore intended to be embracedtherein.

What is claimed is:
 1. A compound of formula (I)

or a pharmaceutically acceptable salt thereof, wherein: A is selectedfrom a bond,

wherein:

denotes the point of attachment to the carbonyl group and

denotes the point of attachment to the nitrogen atom; z is 0, 1, or 2; wis 1 or 2; n is 0 or 1; m is 1 or 2; m′ is 0 or 1; p is 0, 1, or 2;R^(x) is selected from hydrogen, amino, hydroxy, and methyl; R¹⁴ and R¹⁵are independently selected from hydrogen and methyl; and R^(z) isselected from hydrogen and —C(O)NHR¹⁶; wherein R¹⁶ is selected fromhydrogen, —CHR¹⁷C(O)NH₂, —CHR¹⁷C(O)NHCHR¹⁸C(O)NH₂, and—CHR¹⁷C(O)NHCHR¹⁸C(O)NHCH₂C(O)NH₂; wherein R¹⁷ is selected from hydrogenand —CH₂OH and wherein R¹⁸ is selected from hydrogen and methyl; R^(v)is hydrogen or a natural amino acid side chain; R^(c), R^(f), R^(h),R^(i), and R^(m) are hydrogen; R^(n) is hydrogen or methyl or R^(v) andR^(n) form a pyrrolidine ring; R^(a) is hydrogen or methyl; R^(j) isselected from hydrogen, C₁-C₆alkoxyC₁-C₆alkyl, C₁-C₆alkyl,carboxyC₁-C₆alkyl, haloC₁-C₆alkyl, hydroxyC₁-C₆alkyl,(NR^(a′)R^(b′))C₁-C₆alkyl wherein R^(a′) and R^(b′) are independentlyselected from hydrogen and C₁-C₆alkyl; and phenylC₁-C₆alkyl wherein thephenyl is optionally substituted with one, two, three, four or fivegroups independently selected from C₁-C₆alkoxy, C₁-C₆alkyl, cyano, halo,and nitro; R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², and R¹³are independently selected from a natural amino acid side chain and anunnatural amino acid side chain; or R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹,R¹⁰, R¹¹, R¹², and R¹³ can each independently form a ring with thecorresponding vicinal R group as described below; R^(b) is selected fromC₁-C₆alkoxyC₁-C₆alkyl, C₁-C₆alkyl, carboxyC₁-C₆alkyl, haloC₁-C₆alkyl,hydroxyC₁-C₆alkyl, (NR^(a′)R^(b′))C₁-C₆alkyl wherein R^(a′) and R^(b′)are independently selected from hydrogen and C₁-C₆alkyl; andphenylC₁-C₆alkyl wherein the phenyl is optionally substituted with one,two, three, four or five groups independently selected from C₁-C₆alkoxy,C₁-C₆alkyl, cyano, halo, and nitro; or, R^(b) and R², together with theatoms to which they are attached, form a ring selected from azetidine,pyrolidine, morpholine, piperidine, piperazine, and tetrahydrothiazole;wherein each ring is optionally substituted with one to four groupsindependently selected from amino, cyano, methyl, halo, and hydroxy;R^(d) is selected from hydrogen, C₁-C₆alkoxyC₁-C₆alkyl, C₁-C₆alkyl,carboxyC₁-C₆alkyl, haloC₁-C₆alkyl, hydroxyC₁-C₆alkyl,(NR^(a′)R^(b′))C₁-C₆alkyl wherein R^(a′) and R^(b′) are independentlyselected from hydrogen and C₁-C₆alkyl; and phenylC₁-C₆alkyl wherein thephenyl is optionally substituted with one, two, three, four or fivegroups independently selected from C₁-C₆alkoxy, C₁-C₆alkyl, cyano, halo,and nitro; or, R^(d) and R⁴, together with the atoms to which they areattached, can form a ring selected from azetidine, pyrolidine,morpholine, piperidine, piperazine, and tetrahydrothiazole; wherein eachring is optionally substituted with one to four groups independentlyselected from amino, cyano, methyl, halo, hydroxy, and phenyl; R^(e) isselected from hydrogen, C₁-C₆alkoxyC₁-C₆alkyl, C₁-C₆alkyl,carboxyC₁-C₆alkyl, haloC₁-C₆alkyl, hydroxyC₁-C₆alkyl,(NR^(a′)R^(b′))C₁-C₆alkyl wherein R^(a′) and R^(b′) are independentlyselected from hydrogen and C₁-C₆alkyl; and phenylC₁-C₆alkyl wherein thephenyl is optionally substituted with one, two, three, four or fivegroups independently selected from C₁-C₆alkoxy, C₁-C₆alkyl, cyano, halo,and nitro; or R^(e) and R⁵′ together with the atoms to which they areattached, can form a ring selected from azetidine, pyrolidine,morpholine, piperidine, piperazine, and tetrahydrothiazole; wherein eachring is optionally substituted with one to four groups independentlyselected from amino, cyano, methyl, halo, and hydroxy; R^(g) is selectedfrom hydrogen, C₁-C₆alkoxyC₁-C₆alkyl, C₁-C₆alkyl, carboxyC₁-C₆alkyl,haloC₁-C₆alkyl, hydroxyC₁-C₆alkyl, (NR^(a′)R^(b′))C₁-C₆alkyl whereinR^(a′) and R^(b′) are independently selected from hydrogen andC₁-C₆alkyl; and phenylC₁-C₆alkyl wherein the phenyl is optionallysubstituted with one, two, three, four or five groups independentlyselected from C₁-C₆alkoxy, C₁-C₆alkyl, cyano, halo, and nitro; or R^(g)and R⁷, together with the atoms to which they are attached, can form aring selected from azetidine, pyrolidine, morpholine, piperidine,piperazine, and tetrahydrothiazole; wherein each ring is optionallysubstituted with one to four groups independently selected from amino,benzyl optionally substituted with a halo group, benzyloxy, cyano,cyclohexyl, methyl, halo, hydroxy, isoquinolinyloxy optionallysubstituted with a methoxy group, quinolinyloxy optionally substitutedwith a halo group, and tetrazolyl; and wherein the pyrrolidine and thepiperidine ring are optionally fused to a cyclohexyl, phenyl, or indolegroup; provided that at least one of R^(a), R^(b), R^(d), R^(e), R^(g),R^(j), R^(k), and R^(l) is selected from, C₁-C₆alkoxyC₁-C₆alkyl,C₂-C₆alkyl, carboxyC₁-C₆alkyl, haloC₁-C₆alkyl, hydroxyC₁-C₆alkyl,(NR^(a′)R^(b′))C₁-C₆alkyl wherein R^(a′) and R^(b′) are independentlyselected from hydrogen and C₁-C₆alkyl; and phenylC₁-C₆alkyl wherein thephenyl is optionally substituted with one, two, three, four or fivegroups independently selected from C₁-C₆alkoxy, C₁-C₆alkyl, cyano, halo,and nitro.
 2. A compound of claim 1, or a pharmaceutically acceptablesalt thereof, wherein R^(e) is selected from hydrogen and methyl, orR^(e) and R⁵′ together with the atoms to which they are attached, canform a ring selected from azetidine, pyrolidine, morpholine, piperidine,piperazine, and tetrahydrothiazole; wherein each ring is optionallysubstituted with one to four groups independently selected from amino,cyano, methyl, halo, and hydroxy; and R^(j) is hydrogen or methyl.
 3. Acompound of claim 2, or a pharmaceutically acceptable salt thereof,wherein R^(k) is selected from hydrogen and methyl, or R^(k) and R¹¹,together with the atoms to which they are attached, can form a ringselected from azetidine, pyrolidine, morpholine, piperidine, piperazine,and tetrahydrothiazole; wherein each ring is optionally substituted withone to four groups independently selected from amino, cyano, methyl,halo, and hydroxy; and R¹ is methyl, or, R¹ and R¹², together with theatoms to which they are attached, form a ring selected from azetidineand pyrolidine, wherein each ring is optionally substituted with one tofour groups independently selected from amino, cyano, methyl, halo, andhydroxy.
 4. A compound of claim 3, or a pharmaceutically acceptable saltthereof, wherein A is

wherein z and w are each 1; R¹⁴ and R¹⁵ are hydrogen; and R^(z) is—C(O)NHR¹⁶; wherein R¹⁶ is —CHR¹⁷C(O)NH₂; R¹⁷ is hydrogen; R¹ is benzyloptionally substituted with hydroxy; R² is hydrogen or methyl; R⁸ is—(CH₂)indolyl; R¹⁰ is selected from —(CH₂)indolyl and—(CH₂)benzothienyl, each optionally substituted with —CH₂CO₂H; R¹¹ isbutyl; and R¹² is butyl.
 5. A compound selected from: Example 3003,Example 3004, Example 3005, Example 3006, Example 3007, Example 3008,Example 3009, Example 3010, Example 3011, Example 3012, Example 3013,Example 3014, Example 3015, Example 3016, Example 3017, Example 3019,Example 3020, Example 3021, Example 3022, Example 3023, Example 3024,Example 3025, Example 3026, Example 3028, Example 3029, Example 3030,Example 3032, Example 3033, Example 3037, Example 3038, Example 3041,Example 3044, Example 3045, Example 3046, Example 3047, Example 5001,Example 5002, and Example 5003; or a pharmaceutically acceptable saltthereof.
 6. A method of enhancing, stimulating, and/or increasing theimmune response in a subject in need thereof, said method comprisingadministering to the subject a therapeutically effective amount of acompound of claim 1 or a therapeutically acceptable salt thereof.
 7. Themethod of claim 6 further comprising administering an additional agentprior to, after, or simultaneously with the compound of claim 1 or atherapeutically acceptable salt thereof.
 8. The method of claim 7wherein the additional agent is an antimicrobial agent, an antiviralagent, a cytotoxic agent, and/or an immune response modifier.
 9. Themethod of claim 7 wherein the additional agent is an HDAC inhibitor. 10.The method of claim 7 wherein the additional agent is a TLR7 and/or TLR8agonist.
 11. A method of inhibiting growth, proliferation, or metastasisof cancer cells in a subject in need thereof, said method comprisingadministering to the subject a therapeutically effective amount acompound of claim 1 or a therapeutically acceptable salt thereof. 12.The method of claim 11 wherein the cancer is selected from melanoma,renal cell carcinoma, squamous non-small cell lung cancer (NSCLC),non-squamous NSCLC, colorectal cancer, castration-resistant prostatecancer, ovarian cancer, gastric cancer, hepatocellular carcinoma,pancreatic carcinoma, squamous cell carcinoma of the head and neck,carcinomas of the esophagus, gastrointestinal tract and breast, andhematological malignancies.
 13. A method of treating an infectiousdisease in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of acompound of claim 1 or a therapeutically acceptable salt thereof. 14.The method of claim 13 wherein the infectious disease is caused by avirus.
 15. The method of claim 14 wherein the virus is selected fromHIV, Hepatitis A, Hepatitis B, Hepatitis C, herpes viruses, andinfluenza.
 16. A method of treating septic shock in a subject in needthereof, the method comprising administering to the subject atherapeutically effective amount of a compound of claim 1 or atherapeutically acceptable salt thereof.
 17. A method blocking theinteraction of PD-L1 with PD-1 and/or CD80 in a subject, said methodcomprising administering to the subject a therapeutically effectiveamount of a compound of claim 1 or a therapeutically acceptable saltthereof.